Markers of tumor cell response to anti-cancer therapy

ABSTRACT

Compositions and methods for determining circulating biomolecules before, during, and/or after treatment of a patient with an anti-cancer or anti-tumor drug (or putative drug) are described. Methods of treatments based on the compositions and methods described herein are also provided. Noninvasive methods and kits are provided for assessing the efficacy of an anti-cancer therapy for killing or damaging cancer cells. Embodiments are used to determine the cancer-killing efficacy of an anti-cancer drug in a patient, to optimize the selection of an anti-cancer drug for treatment of a patient, to adjust the dosage of an anti-cancer drug for treatment of a particular cancer in a patient and for identifying useful anti-cancer therapeutics for any one particular type of cancer.

TECHNICAL FIELD

Methods provided herein are useful in the field of cancer and cancertherapeutics, and in particular, in determining efficacy of cancertherapeutics. Methods and compositions for measuring circulatingbiomolecules such as, e.g., nucleic acids and proteins, in conjunctionwith anti-cancer therapy.

BACKGROUND

The term cancer commonly refers to a broad group of diseasescharacterized by unregulated cell growth that forms malignant tumors.Cancer therapies generally kill cells, ideally primarily tumor cells butalso normal cells. Current methods for determining the effectiveness ofcancer therapy include invasive procedures such as biopsies, as well asimaging methods such as CT scan, magnetic resonance imaging (MRI) scan,and positron emissions tomography (PET) scan. However, to take advantageof such non-invasive methods, the tumors typically must reduce in sizeenough that the imaging procedures can detect a difference. To aid indetermining whether a new anti-cancer drug or anti-tumor drug hasefficacy in killing cancer or tumor cells, or whether an anti-cancerdrug or anti-tumor drug has efficacy against a particular tumor orcancer, it would be very useful to have a method to determinenon-invasively the amount of tumor or cancer cell killing in a patientrelative to normal cell killing. Methods and compositions fornon-invasive determination of circulating biomolecules following tumoror cancer cell killing by a therapeutic or putative therapeutic are alsodesirable.

MicroRNAs are small (approximately 22 nucleotide) single-stranded RNAsfound predominantly in the cytoplasm of higher eukaryotes (plants andmulti-cellular animals). Their primary function is to regulate geneexpression by binding to specific target mRNAs, usually in the3′-untranscribed region (3′-UTR), and inhibiting their translation whilepromoting their destruction. There are over 1,000 identified miRNAs inmouse and over 2,000 in humans. Most miRNAs are thought to have multiplemRNA targets that could number in the hundreds. Many mRNAs that areregulated by miRNAs have binding sites in their 3′-UTRs for multiplemiRNAs. Some miRNAs are fairly ubiquitously expressed; others exhibithighly restricted tissue-specific expression.

Recent research has discovered that a subset of a few hundred miRNAs ispresent and readily detectable in the serum and plasma of mammals. Theprofile of the serum/plasma miRNAs is remarkably stable in normalhealthy animals, but can vary in disease states or in response to drugsor chemically induced toxicities. The serum/plasma miRNA profile differsfrom that for the cellular component of the blood.

A growing literature is beginning to reveal many examples ofserum/plasma miRNA profiles that provide biomarkers for diagnosis andprognosis in diseases such as liver fibrosis, myocardial malfunction,and various cancers. Serum/plasma miRNA profiling has also been shown topredict drug efficacy, toxicity, and specific organ and tissue damage.

SUMMARY

In various aspects, compositions and methods for determining circulatingbiomolecules before, during, and/or after treatment of a patient with ananti-cancer or anti-tumor drug (or putative drug) are described. Methodsof treatments based on the compositions and methods described herein arealso provided. Aspects and embodiments that are directed to anti-cancertherapy, whether expressly stated or not, are aspects and embodimentsthat may be directed to anti-tumor therapies as well. Similarly, aspectsand embodiments that employ anti-cancer drugs may also be employed withanti-tumor drugs.

In various aspects, noninvasive methods and kits are provided forassessing the efficacy of an anti-cancer therapy for killing or damagingcancer cells. Embodiments are used to determine the cancer-killingefficacy of an anti-cancer drug in a patient, to optimize the selectionof an anti-cancer drug for treatment of a patient, to adjust the dosageof an anti-cancer drug for treatment of a particular cancer in a patientand for identifying useful anti-cancer therapeutics for any oneparticular type of cancer.

Provided herein are noninvasive methods for determining cancer-killingefficacy in a patient treated with an anti-cancer therapy and inparticular an anti-cancer drug. In some embodiments, the methodscomprise measuring a blood level of an intracellular cancercell-specific marker in the patient following administration of aputative anti-cancer drug. In some embodiments, the level of a tumorcell-specific marker can be measured before and after administration ofan anti-cancer drug. Changes in the level of such a tumor cell-specificmarker can be indicative of efficacy of the putative anti-cancer drug.An increase in the level of the intracellular cancer cell-specificmarker in the blood of the patient compared to a control marker isindicative of efficacy of the putative anti-cancer drug. In otherembodiments, a decrease in the level of the intracellular cancercell-specific marker in a sample compared to a control marker isindicative of efficacy of the putative anti-cancer drug. In certainembodiments, changes in the level of a tumor cell-specific markercorrelates with therapeutic efficacy of the anti-tumor drug.

The patient can be treated with the putative anti-cancer drug if thelevel of intracellular cancer cell-specific marker in the patient'scirculation increases following administration of the putativeanti-cancer drug in relation to the control marker. Alternatively,patients can be treated with the putative anti-cancer drug if the levelof cancer cell-specific marker in a sample decreases followingadministration of the putative anti-cancer drug in relation to thecontrol marker. In some embodiments, the anti-cancer drug is ananti-tumor drug conjugate of an antigen-binding protein and a drug, thecancer cell-specific marker is a tumor-specific marker, and the canceris a solid tumor or a B-cell related cancer. In certain embodiments, thecancer cell-specific marker is a micro RNA (miRNA).

Also provided are noninvasive methods of selecting an effectiveanti-cancer drug for treatment of a patient in need thereof. In someembodiments, the methods comprise measuring a first level of a cancercell-specific marker in a first sample from a cancer-bearing patient,administering a putative anti-cancer drug to the patient, and measuringa second level of the cancer cell-specific marker in a second samplefrom the patient. An increase in the second level indicates anti-cancerefficacy of the putative anti-cancer drug and the patient is treatedwith the putative anti-cancer drug, thereby selecting an effectiveanti-cancer drug for treatment of the patient. In some embodiments, theincrease in the level of the cancer cell-specific marker in the blood ofthe patient is compared to a corresponding change in a control marker.Depending on the marker selected, a decrease in the second level canindicate efficacy of the putative anti-cancer drug such that patientsare treated with the putative anti-cancer drug, thereby selecting aneffective anti-cancer drug for treatment of the patient. In certainembodiments the marker measured to determine efficacy of the anti-cancerdrug is a tumor-responsive biomarker. In some embodiments, theanti-cancer drug is an anti-tumor drug conjugate of an antigen-bindingprotein and a drug, the cancer cell-specific marker is a tumor-specificmarker, and the cancer is a solid tumor or a B-cell related cancer. Inone embodiment, a control marker is a marker from a non-cancer (ornon-tumor) cell.

Further provided are noninvasive methods of adjusting a dosage of ananti-cancer drug for treatment of a cancer in a patient. In someembodiments, the methods comprise measuring a level of one or morecancer cell-specific markers in the circulation of a patient who hasbeen administered an initial amount of the anti-cancer drug, andadjusting the dosage for subsequent administration of the anti-cancerdrug to the patient based upon the level of the one or more cancercell-specific markers in the circulation of the patient afteradministration of the initial amount of the anti-cancer drug. In someembodiments, the methods further comprise measuring at least one controlmarker in the circulation for normalization of the level of the one ormore cancer cell-specific markers. In certain embodiments the markermeasured to determine efficacy of the anti-cancer drug is atumor-responsive biomarker. In some embodiments, the anti-cancer drug isan anti-tumor drug conjugate of an antigen-binding protein and a drug,the cancer cell-specific marker is a tumor-specific marker, and thecancer is a solid tumor or a B-cell related cancer.

For example, patients having a higher level of a tumor-responsivebiomarker after the first administration of the anti-tumor drug could,on average, receive a greater second dose of the anti-tumor drug thanpatients having a lower level of the tumor-responsive biomarker afterthe first dose of the anti-tumor drug. Alternatively, depending on theindividual marker, patients having a lower level of the tumor-responsivebiomarker after the first administration of the anti-tumor drug could,on average, receive a greater second dose of the anti-tumor drug thanpatients having a higher level of the tumor-responsive biomarker afterthe first dose of the anti-tumor drug.

Still further provided are noninvasive methods for assessing atherapeutic efficacy of an anti-cancer drug in a patient. In someembodiments, the methods comprise (a) obtaining a first baseline samplefrom the patient's circulation, wherein the patient has cancer (or atumor); (b) administering to the patient a dose of an anti-cancer drug,wherein at least one cancer cell-specific marker is sequestered withinthe cancer cell of the patient prior to administration of theanti-cancer drug but is released into the patient's circulationfollowing administration of the anti-cancer drug; (c) obtaining a secondsample from the patient's circulation; (d) measuring an amount of the atleast one cancer cell-specific marker (i) in the first baseline sampleand (ii) in the second sample, and (e) comparing the amount of thecancer cell-specific marker in the first baseline sample with the amountof the cancer cell-specific marker in the second sample. An increase inthe amount of the at least one cancer cell-specific marker in the secondsample relative to the amount of the at least one cancer cell-specificmarker in the first baseline sample is indicative of increased cancercell death in the patient. The patient can be treated with theanti-cancer drug that increases the at least one cancer cell-specificmarker. Alternatively, depending on the individual marker, patientshaving a lower level of the biomarker after the first administration ofthe anti-tumor drug could, on average, receive a greater second dose ofthe anti-tumor drug than patients having a higher level of the biomarkerafter the first dose of the anti-tumor drug. In some embodiments, theanti-cancer drug is an anti-cancer drug conjugate of an antigen-bindingprotein and a drug, the cancer cell-specific marker is a tumor-specificmarker, and the cancer is a solid tumor or a B-cell related cancer. Alsoprovided herein are tumor-responsive biomarkers that can change inresponse to the tumor burden, but are not necessarily derived from thetumor.

It is also contemplated that embodiments herein can be used to identifyanti-cancer drugs with high efficacy on particular types of cancer. Inparticular, a subject, typically a mouse or other rodent having apre-determined form of cancer, is tested for a baseline level of one ormore biomolecules associated with the pre-determined form of cancer,e.g., a CD20 marker for lymphoma. The proposed anti-cancer drug for thepre-determined form of cancer is administered to the subject and thelevel of the biomolecule determined. Increases or decreases of thebiomolecule compared to control markers are used to identify anti-cancerdrugs with greater potency for killing or damaging the pre-determinedform of cancer in the subject. Alternatively, increases or decreases ofthe biomolecule before and after administration of the anti-cancer drugcan be used to identify candidate anti-cancer drugs with greater potencyfor killing or damaging the pre-determined form of cancer cells in thesubject. It is contemplated that a plurality of anti-cancer drugs can betested using this method to identify and screen for anti-cancer drugshaving enhanced efficacy for various forms of cancer. In addition,anti-cancer drugs having greater efficacy for one type of cancer overanother type of cancer can also be identified, thereby maximizing cancercell killing and damage, for any one anti-cancer drug.

In one aspect, a method for selecting an anti-cancer drug for treatmentof a human tumor is provided, comprising introducing into a suitablehost non-human animal (e.g., any rodent or mouse) a xenograft of thehuman tumor, administering a putative or candidate anti-cancer agent tothe xenografted host non-human animal (e.g., a mouse), and determiningthe level of one or more intracellular tumor markers that have enteredthe mouse's circulation from a cell of the xenograft, wherein a putativeanti-tumor agent that releases a predetermined level of one or more ofthe intracellular tumor cell markers from the cell of the xenograft isselected as a suitable therapeutic for treating the human tumor. In someembodiments, a tumor cell, a tumor tissue, or a tumor organ can beintroduced onto any host animal, (e.g., any rodent) in order todetermine the effectiveness of an anti-tumor drug by measuring the levelof a tumor-cell specific marker before and after administration of theanti-tumor drug.

In another embodiment, a putative or candidate anti-cancer drug isselected based on the level of a cell-specific marker before and afteradministration of a drug to a host non-human animal (e.g., any rodent)xenografted with a tumor cell, a tumor tissue, or a tumor organ. Forexample, in some embodiments, an anti-cancer drug is selected whereinthe level of a tumor cell-specific marker is decreased followingadministration of the putative anti-tumor agent to a xenografted hostnon-human animal (e.g., any rodent) compared to the level of tumorcell-specific marker prior to administration of the candidateanti-cancer agent. In other embodiments, an anti-cancer drug is selectedwherein the level of a tumor cell-specific marker is increased followingadministration of the putative or candidate anti-tumor drug to thexenografted host non-human animal (e.g., any rodent) compared to thelevel of the tumor cell-specific marker prior to administration of theanti-cancer agent. In certain embodiments, the marker is a miRNA markerand the tumor is lung cancer, prostate cancer, or colon cancer.

In one aspect, a method for selecting an anti-cancer drug for treatmentof a patient that has a tumor is provided, comprising introducing into asuitable mouse or host non-human animal (e.g., any rodent) a xenograftof the patient's tumor, administering a putative or candidateanti-cancer agent to the xenografted mouse or host non-human animal(e.g., any rodent), and determining the level of one or moreintracellular tumor markers that have entered the mouse's circulationfrom a cell of the xenograft, wherein a putative or candidate anti-tumoragent that releases a predetermined level of one or more of theintracellular tumor cell markers from the cell of the xenograft isselected as a suitable therapeutic for treating the patient. Thus, asused herein the term “derived from” refers to a tumor cell, a tumortissue, or a tumor organ taken from a human patient or propagated from atumor cell or tumor tissue taken from a human patient.

Embodiments herein provide kits for qualifying cancer status in asubject, wherein the kits can be used to detect the differentialpresence of the biomarkers described herein. For example, the kits canbe used to detect a differential presence of any combination of thebiomarkers in tumor samples of cancer subjects before and after exposureto an anti-cancer drug or other therapeutic drug. The kits of theinvention have many applications. For example, the kits can be used tomonitor efficacy of a therapeutic drug in a cancer subject. The kits canalso be used to identify agents useful in the treatment of cancer.

Other features and advantages of the disclosure will be apparent fromthe following description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the increase in certain miRNA markers upon A549 lung cancertumor implantation and subsequent decrease in miRNA marker level threedays following administration of the anti-tumor drug Cisplatin.

FIG. 2 shows that particular miRNA marker levels decrease uponadministration of Cisplatin to lung tumors (A549) and administration ofDocetaxel to prostate tumors (PC3M).

FIG. 3 shows the decrease in certain miRNA markers upon A549 lung cancertumor implantation and subsequent increase in miRNA marker level threedays following administration of the anti-tumor drug Cisplatin.

FIG. 4 shows the decrease in miR-16-5p, miR-1208, miR-24-3p, and miR-588upon A549 lung cancer tumor implantation and subsequent increase inmiRNA marker level three days following administration of the anti-tumordrug Cisplatin.

FIG. 5 shows the increase in certain miRNA markers upon PC3M prostatecancer tumor implantation and subsequent decrease in miRNA marker levelfollowing administration of the anti-tumor drug Docetaxel.

FIG. 6 shows the increase in miR-190, miR-153, and miR-92a-1 upon PC3Mprostate cancer tumor implantation and subsequent decrease in miRNAmarker level following administration of the anti-tumor drug Docetaxel.

FIG. 7 shows the decrease in certain miRNA markers upon PC3M prostatecancer tumor implantation and subsequent increase in miRNA marker levelfollowing administration of the anti-tumor drug Docetaxel.

FIG. 8 shows the decrease in miR-634, and miR-647 upon PC3M prostatecancer tumor implantation and subsequent increase in miRNA marker levelfollowing administration of the anti-tumor drug Docetaxel.

DETAILED DESCRIPTION

Provided herein are methods, which determine cancer-killing (ortumor-killing) efficacy of an anti-cancer (or anti-tumor drug),including in a patient treated with an anti-cancer drug, methods ofselecting an effective anti-cancer drug for treatment in a patient inneed thereof, methods of adjusting a dosage of an anti-cancer drug fortreatment of a cancer in a patient, and methods of assessing atherapeutic efficacy of an anti-cancer drug in a patient. Each of thesemethods is noninvasive, requiring the patient to provide samples thatcan be obtained from a simple blood test or from blood, plasma, serum,cerebrospinal fluid, synovial fluid, lymph, saliva, or urine obtainedfrom a subject. These methods provide fast and accurate information tothe patient and health care professional thereby providing andmaximizing the usefulness of treatments reliant on anti-cancer drugs orother anti-cancer therapeutics. Methods are also provided for screeningand selecting anti-cancer therapeutics for target cancers, andidentifying target therapeutics most useful in treating any oneparticular type of cancer.

In this light, methods are disclosed, which measure the levels ofcertain cancer-specific biomarkers, such as microRNAs, cell free DNA(cfDNA), and other cytosolic or nuclear macromolecules normallycontained within cells, but that are released from cancer cells afterexposure to an anti-cancer drug; and compare the release of controlmarkers from normal cells after exposure to the same anti-cancer drug todetermine the extent and ratio of cancer cells killed relative to normalcells killed following the anti-cancer treatment. In particularembodiments, the biomarkers are tumor-responsive biomarkers that respondto changes in tumor burden but are not necessarily derived from a tumorcell, tumor tissue, or tumor organ. In some circumstances, the levels ofcertain biomarkers (e.g., miRNA markers) in a sample are decreasedfollowing administration of anti-cancer drug.

Before embodiments of the present invention are further described, it isto be understood that methods described herein are not limited by therecited experimental conditions; as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the various embodiments of the methods and materials are nowdescribed. All patents, applications and non-patent publicationsmentioned in this specification are incorporated herein by reference intheir entireties.

Unless defined otherwise, all technical and scientific terms used hereininclude the meaning commonly understood by a person skilled in the artto which this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Lackie and Dow, The Dictionary of Cell & Molecular Biology (3ed. 1999); Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used herein, the term “about”, when used in reference to a particularrecited numerical value, means that the value may vary from the recitedvalue by no more than 1%. For example, as used herein, the expression“about 100” includes 99 and 101 and all values in between (e.g., 99.1,99.2, 99.3, 99.4, etc.).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, lymphoma and leukemia, and solid tumors. By “B cell-related cancer”or “cancer of B-cell lineage” or “neoplastic B-cell growth” is intendedany type of cancer in which the dysregulated or unregulated cell growthis associated with B cells.

“Tumor” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. “Neoplastic” as used herein, refers to anyform of dysregulated or unregulated cell growth, whether malignant orbenign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth. As described herein, the compositions and methods can beused for both anti-cancer and anti-tumor applications.

Cancer can be from an organ, e.g., selected from the group consisting ofskin, colon, thyroid, ovarian, lung, and pancreas. In one embodiment,the skin tumor is a melanoma. In another embodiment, the tumor isselected from the group consisting of: prostate carcinoma, lungcarcinoma, breast carcinoma, ovarian carcinoma, skin carcinoma, coloncarcinoma, urinary bladder carcinoma, liver carcinoma, gastriccarcinoma, renal cell carcinoma, nasopharyngeal carcinoma, squamous cellcarcinoma, thyroid papillary carcinoma, cervical carcinoma, sarcomas,glioma, acute myelogenous leukemia, pancreatic carcinoma, and head andneck carcinomas. In yet another embodiment, the cancer is non-Hodgkin'slymphoma, chronic lymphocytic leukemia, multiple myeloma, B celllymphoma, high-grade B cell lymphoma, intermediate-grade B celllymphoma, low-grade B cell lymphoma, B cell acute lympohoblasticleukemia, Hodgkin's disease, plasmacytoma, follicular lymphoma,follicular small cleaved lymphoma, follicular large cell lymphoma,follicular mixed small cleaved lymphoma, diffuse small cleaved celllymphoma, diffuse small lymphocytic lymphoma, prolymphocytic leukemia,lymphoplasmacytic lymphoma, marginal zone lymphoma, mucosal associatedlymphoid tissue lymphoma, monocytoid B cell lymphoma, splenic lymphoma,hairy cell leukemia, diffuse large cell lymphoma, mediastinal large Bcell lymphoma, lymphomatoid granulomatosis, intravascular lymphomatosis,diffuse mixed cell lymphoma, diffuse large cell lymphoma, immunoblasticlymphoma, Burkitt's lymphoma, AIDS-related lymphoma, Waldenstrom'sMacroglobulinemia, mantle cell lymphoma, and heavy chain disease. Insome aspects, the tumor is characterized by neoplastic B-cell growth.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to anantigen-binding protein or an antigen-binding fragment thereof so as togenerate a “labeled” antigen-binding protein or an antigen-bindingfragment thereof. The label may be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

A “subject” is a vertebrate, for example, a mammal, and illustratively,a primate such as a human. Mammals include, but are not limited to,primates, (including humans), farm animals, sport animals, wildlife, andpets. A “patient” can be any subject but is typically a human. A“patient” can also refer to a plurality of patients, such as a pluralityof human patients.

A “sample” or “biological sample” includes, for example, blood, plasma,serum, cerebrospinal fluid, synovial fluid, lymph, saliva, or urineobtained from a subject. A sample can be any fluid or component obtainedfrom a subject in which the level of a cell-specific marker can bemeasured.

“Tumor cell-specific marker” as used herein includes a polypeptide (of aparticular molecular weight) or nucleic acid, which is sequesteredwithin a tumor cell prior to death of the tumor cell, e.g., prior totreatment with an anti-tumor drug but is released into the patient'scirculation following the treatment. A “tumor cell-specific marker” alsoincludes markers that are released by tumor cells prior to damage ordeath of the tumor cell (e.g., prior to treatment with an anti-tumordrug) but are maintained in the cell and not following the treatmentwith an anti-tumor drug. In certain embodiments, tumor cell-specificmarkers are miRNA markers. Polypeptide biomarkers can be identified bymolecular mass in Daltons, and include the masses centered on theidentified molecular masses for each marker. Nucleic acid biomarkers canbe identified by sequence. “Tumor cell-specific biomarker” includes, forexample, biomarkers of cellular apoptosis, cell proliferation andsurvival. Exemplary tumor-specific biomarkers include, but are notlimited to, CD20, B lymphoid tyrosine kinase (BLK), and combinationsthereof. In various aspects, cancer or tumor cells may diespontaneously, or for natural or unknown reasons, and in variousembodiments a change in the level of a particular marker in response toanti-cancer or anti-tumor therapy is an indicator of efficacy orputative efficacy of the anti-cancer or anti-tumor therapy in a human.

The term “biomarker” as used herein includes, but is not limited to, anucleic acid, peptide, protein, lipid, antigen, carbohydrate orproteoglycan, such as DNA (including, for example, cell-free DNA(cfDNA)) or RNA. The RNA can be mRNA, miRNA, snoRNA, snRNA, rRNAs,tRNAs, siRNA, hnRNA, or shRNA, or short or long non-coding RNAs. The DNA(e.g., cfDNA) or RNA (e.g., mRNA) can include point mutations, DNAhypermethylations, microsatellite instabilities, and losses ofheterozygosity, or a combination thereof.

The term “tumor-responsive biomarker” as used herein includes a normalcomponent of the serum, plasma, or other body fluid that changes inresponse to tumor burden but is not necessarily derived from the tumor.The tumor-responsive biomarkers may be either directly or inverselyproportional to the tumor burden. That is, the level of thetumor-responsive biomarker may increase as the tumor burden decreases,or the level of the tumor-responsive biomarker may decrease as the tumorburden decreases. As used herein, the term “tumor burden” refers to theamount of tumors in a patient or non-human animal. In some embodiments atumor-responsive biomarker includes a tumor cell-specific marker. In oneembodiment, the tumor-responsive biomarker is a tumor type-specificbiomarker. For example, the tumor-responsive biomarker can be specificfor lung tumors, prostate tumors, or colon tumors. In one embodiment,the tumor-responsive biomarker is a tumor cell-specific biomarker. Inone embodiment, the tumor-responsive biomarker is an anti-cancerdrug-specific biomarker. For example, the tumor-responsive biomarker canbe specific for treatments with certain types of drugs. In specificembodiments, the tumor-responsive biomarker is a miRNA marker.

Particularly useful biomarkers include those typically not secreted,e.g. mRNAs, rRNA, microRNAs, DNAs, and a combination thereof. In someembodiments, the biomarker is an intracellular biomarker, e.g., one ormore intracellular proteins. In one embodiment, the intracellularproteins are cytosolic protein. In one embodiment, the biomarker is atransmembrane or membrane-associated protein. In one embodiment, theintracellular proteins are organellar proteins residing in or associatedwith an organelle. In one embodiment, the intracellular proteins arenuclear proteins. In certain embodiments, useful biomarkers for themethods disclosed herein are released by tumor cells into theextracellular space, into the blood, or any other area surrounding thecells prior to treatment with an effective anti-tumor drug, but are notreleased following administration of the anti-tumor drug.

“Non-specific” or “control” marker can be a general marker of celltoxicity, markers, which are not specific to tumor cells.

The term “measuring” includes methods, which include determining,detecting, or observing the presence or absence of marker(s) in thesample, quantifying the amount of marker(s) in the sample, and/orqualifying the type of biomarker (e.g., measuring epigenetic changes,sequence changes, etc.). Measuring can be accomplished by methods knownin the art and those further described herein. Any suitable methods canbe used to detect and measure one or more of the markers describedherein. These methods include, without limitation, immunoassays, massspectrometry (e.g., laser desorption/ionization mass spectrometry,SELDI), fluorescence (e.g., sandwich immunoassay), surface plasmonresonance, ellipsometry, atomic force microscopy, PCR (includingquantitative PCR, e.g., real-time PCR), and microarray analysis (forexample, with Significance Analysis of Microarrays (SAM) software). Inone embodiment, microarray analysis is used to detect microRNA, known asmicroRNA or miRNA expression profiling.

In some embodiments, a difference in the amount of the tumorcell-specific marker in the sample as compared to a control or baselineindicates that the putative anti-tumor drug has a therapeutic efficacy.For example, an increase (e.g., a one-fold, a two-fold, a three-fold, afour-fold, a five-fold, a six-fold, a seven-fold, an eight-fold, anine-fold, or a ten-fold or more increase) in the amount of a marker inthe sample as compared to the control indicates that the putativeanti-tumor drug has efficacy. That is, the change in the amount of thetumor cell-specific marker in the sample can be at least one fold (e.g.,a two-fold, a three-fold, a four-fold, a five-fold, a six-fold, aseven-fold, an eight-fold, a nine-fold, or a ten-fold or more) higherthan the change in the amount of control marker. In some embodiments,the change in the amount of the tumor cell-specific marker in the samplecan be at least one fold (e.g., a two-fold, a three-fold, a four-fold, afive-fold, a six-fold, a seven-fold, an eight-fold, a nine-fold, or aten-fold or more) lower than the change in the amount of control marker.In certain embodiments, the marker is a tumor-responsive biomarker.

The change in the amount of the tumor-responsive biomarker can be atleast one fold, at least two fold, at least three fold, at least fourfold, at least five fold, at least six fold, at least seven fold, atleast eight fold, at least nine fold, at least ten fold or more in asample obtained before administration of an anti-tumor drug compared toa sample obtained after administration of an anti-tumor drug. The changein the amount of tumor-responsive biomarker before and after theadministration of an anti-tumor drug can be an increase or a decrease.

In certain embodiments, an increase (e.g., a one-fold, a two-fold, athree-fold, a four-fold, a five-fold, a six-fold, a seven-fold, aneight-fold, a nine-fold, or a ten-fold or more increase) in the amountof a biomarker after administration of an anti-tumor drug as compared tothe amount of a biomarker before anti-tumor drug administrationindicates that the putative anti-tumor drug has efficacy. In otherembodiments, a decrease, (e.g., a one-fold, a two-fold, a three-fold, afour-fold, a five-fold, a six-fold, a seven-fold, an eight-fold, anine-fold, or a ten-fold or more decrease) in the amount of a biomarkerafter administration of an anti-tumor drug as compared to the amount ofa biomarker before anti-tumor drug administration indicates that theputative anti-tumor drug has efficacy. Likewise, depending on theselected marker, absence of an increase or an absence of a decrease inthe level of a tumor-responsive biomarker after administration of ananti-tumor drug can indicate that the administration of the anti-cancerdrug was not therapeutically effective.

While embodiments described herein reflect fold increases over thecontrol, it is further contemplated that certain markers such as tumorsuppressor markers or anti-apoptotic markers may show fold decreasesrelative to the amount of control marker. For example, a decrease (e.g.,a one-fold, a two-fold, a three-fold, a four-fold, a five-fold, asix-fold, a seven-fold, an eight-fold, a nine-fold, or a ten-fold ormore decrease) in the amount of a marker in the sample as compared theamount of control marker can indicate that the putative anti-tumor drughas efficacy. That is, the decreased amount of the marker peptide in thesample can be at least one fold (e.g., a two-fold, a three-fold, afour-fold, a five-fold, a six-fold, a seven-fold, an eight-fold, anine-fold, or a ten-fold or more) lower than the decrease of the amountof control marker.

“Detect” includes identifying the presence, absence or amount of theobject to be detected.

The terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably herein to include a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide”, “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

While the methods described herein are specific to cancer and tumors, itis contemplated herein that the methods are also useful in other diseasestates, including, for example, multiple sclerosis and other autoimmunediseases.

Methods

In various aspects, provided herein are noninvasive methods fordetermining tumor cell-killing efficacy in a patient treated with ananti-tumor drug. In some embodiments, the methods comprise measuring ablood level of an intracellular tumor cell-specific marker in thepatient following administration of a putative anti-tumor drug. Anincrease in the level of the intracellular tumor cell-specific markerfor example, in the blood, plasma, serum, cerebrospinal fluid, synovialfluid, lymph, saliva, or urine of the patient compared to a controlmarker is indicative of efficacy of the putative anti-tumor drug. Insome embodiments, a change in the level of a tumor-cell specific markerafter administration of a putative anti-tumor drug is indicative ofefficacy of the putative anti-tumor drug. Such a change can be anincrease or a decrease in the level of a tumor-cell specific marker. Inspecific embodiments, a change in the level of at least onetumor-responsive biomarker before and after administration of ananti-tumor drug correlates to reduction in the size, severity, and/orprevalence of cancer cells in the patient.

The patient can be treated with the putative anti-tumor drug if thelevel of intracellular tumor cell-specific marker increases followingadministration of the putative anti-tumor drug, particularly if theincrease is relative to the control marker. Patients can also be treatedwith the putative anti-tumor drug if the level of intracellular tumorcell-specific marker decreases following administration of the putativeanti-tumor drug, and/or if the decrease is relative to the controlmarker. In some embodiments the marker used for determining drugefficacy is a tumor-responsive biomarker. In some embodiments, theanti-tumor drug is an anti-tumor drug conjugate of an antigen-bindingprotein and a drug, the tumor cell-specific marker is a cancer-specificmarker, and the cancer is a solid tumor or a B-cell related cancer. Forexample, in certain embodiments, the cancer is prostate, lung, or coloncancer and the marker is a miRNA marker.

As noted throughout, any of the methods described herein can compriseone or more further steps of obtaining levels of a second marker, e.g.,a control (general) cell marker or non-specific marker, which whenpresent in a sample or in circulation indicates damage to normal cells.Thus, the release of control cell markers from normal cells afterexposure to an anti-cancer drug will indicate the level of damage theanti-cancer drug causes to normal tissue and can, in some aspects, beused to provide a therapeutic efficacy of the drug on the tumor relativeto the toxicity of the drug to normal tissue.

Also provided are methods of selecting an effective anti-tumor drug fortreatment of a patient in need thereof. In some embodiments, the methodscomprise measuring a first level of a tumor cell-specific marker in afirst sample from a tumor-bearing patient, administering a putativeanti-tumor drug to the patient, and measuring a second level of thetumor cell-specific marker in a second sample from the patient. Incertain embodiments, the marker is a tumor-responsive biomarker. Anincrease in the second level indicates anti-tumor efficacy of theputative anti-tumor drug and the patient is treated with the putativeanti-tumor drug, thereby selecting an effective anti-tumor drug fortreatment of the patient. A decrease in the second level may alsoindicate efficacy of the putative anti-tumor drug. Likewise, the absenceof an increase or the absence of a decrease in the level of atumor-responsive biomarker can indicate that the administration of theanti-tumor drug is not therapeutically effective. In some embodimentsthe selection of a tumor cell-specific marker will determine whether anincrease or decrease in the level of tumor cell-specific markerfollowing administration of anti-tumor drug is indicative of anti-tumorefficacy of the administered drug. In some embodiments, the anti-tumordrug is an anti-cancer drug, the tumor cell-specific marker is acancer-specific marker, and the tumor-bearing patient is acancer-bearing patient.

Further provided are methods of adjusting or controlling a dosage of ananti-tumor drug for treatment of a tumor in a patient. In someembodiments, the methods comprise measuring a level of one or more tumorcell-specific markers in the circulation of a patient who has beenadministered an initial amount of the anti-tumor drug, and adjusting thedosage for subsequent administration of the anti-tumor drug to thepatient based upon the level of the one or more tumor cell-specificmarkers in the circulation of the patient after administration of theinitial amount of the anti-tumor drug. In some embodiments, theanti-tumor drug is an anti-cancer drug, and the tumor cell-specificmarker is a cancer-specific marker or a tumor-responsive biomarker.

For example, in some embodiments, the dosing regimen of anti-tumor drugadministered to patients is changed when the level of a tumor-responsivebiomarker after the first dose of the anti-tumor drug does not increasecompared to the level of the tumor-responsive biomarker prior toadministration of the anti-tumor drug. In specific embodiments, thedosing regimen of the anti-tumor drug is changed when the level of atleast one miRNA marker including miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-647,miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof, is not increasedafter administration of an anti-tumor drug compared to prior toanti-tumor drug administration.

In some embodiments, the dosing regimen of anti-tumor drug administeredto a patient is changed when the level of the tumor-responsive biomarkerafter the first dose of the anti-tumor drug does not decrease comparedto the level of the tumor-responsive biomarker prior to administrationof the anti-tumor drug. In specific embodiments, the dosing regimen ofthe anti-tumor drug is changed when the level of at least one miRNAmarker including miR-802, miR-30b-3p, miR-510, miR-622, miR-127-3p,miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p,miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p,miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p,miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p, miR-190, miR-153,miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370, miR-92a-1,miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267, miR-621,miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p, miR-101-5p,miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p, miR-598,miR-206, miR-885-5p, miR-1972, and a combination thereof, is notdecreased after administration of an anti-tumor drug compared to priorto anti-tumor drug administration.

Depending on the outcome of the comparison of tumor-responsive biomarkerlevel prior to and following anti-tumor drug administration, differentpatients may receive different second or subsequent dosages ofanti-tumor drug. As used herein, an alteration or adjustment in thedosage regimen of an anti-tumor drug can be an increase in the dosage ora decrease in the dosage of the anti-tumor drug. For example, anincrease or decrease in the dosage of an anti-tumor drug could be anincrease or decrease in the amount, concentration, duration, orfrequency of the anti-tumor drug administration.

According to certain embodiments of the present invention, multipledoses of an anti-tumor drug may be administered to a subject over adefined time course. Such methods can comprise sequentiallyadministering to a subject multiple doses of an anti-tumor drug. As usedherein, “sequentially administering” means that each dose of anti-tumordrug is administered to the subject at a different point in time, e.g.,on different days separated by a predetermined interval (e.g., hours,days, weeks or months). The present disclosure includes methods whichcomprise sequentially administering to the patient a single initial doseof an anti-tumor drug, followed by one or more subsequent doses of theanti-tumor drug depending on the relative level of at least onetumor-responsive biomarker before and after administration of the firstor a subsequent dosage of the anti-tumor drug.

The terms “initial dose,” and “subsequent dose,” refer to the temporalsequence of administration of the anti-tumor drug. Thus, the “initialdose” or “first dose” is the dose which is administered at the beginningof the treatment regimen; the “second dose” or “subsequent dose” is thedose which is administered after the initial dose. The initial andsubsequent doses may all contain the same amount of anti-tumor drug, butgenerally may differ from one another in terms of frequency ofadministration. In certain embodiments, however, the amount anti-tumordrug contained in the initial and subsequent doses varies from oneanother (e.g., adjusted up or down as appropriate) during the course oftreatment depending on the relative levels of at least onetumor-responsive biomarker before and after administration of theanti-tumor drug.

As used herein, the term “dosage regimen” refers to a drugadministration decision regarding formulation, route of administration,drug dose, dosing interval and treatment duration. Thus, if a dosageregimen of an anti-tumor drug is changed in response to an increase ordecrease in the level of at least tumor cell-specific marker followingadministration of the anti-tumor drug, the formulation, route ofadministration, drug dose, dosing interval, or treatment duration can bechanged. For example, if the level of a tumor cell-specific markerfollowing administration of a dose of an anti-tumor drug does notindicate that the dose was therapeutically effective, the dosage regimencan be changed to increase the frequency, concentration, level, route ofadministration, or duration of treatment of the anti-tumor drug. Incertain embodiments, changing the dosage regimen of the anti-drugincreases the therapeutic efficacy of the anti-tumor drug.

In one embodiment, subsequent doses can be administered 1 to 26 (e.g.,1, 1½, 2, 2 1/2, 3, 3 1/2, 4, 4 1/2, 5, 5 1/2, 6, 6 1/2, 7, 7 1/2, 8, 81/2, 9, 9 1/2, 10, 10 1/2, 11, 11 1/2, 12, 12 1/2, 13, 13 1/2, 14, 141/2, 15, 15 1/2, 16, 16 1/2, 17, 17 1/2, 18, 18 1/2, 19, 19 1/2, 20, 201/2, 21, 21 1/2, 22, 22 1/2, 23, 23 1/2, 24, 24 1/2, 25, 25 1/2, 26, 261/2, or more) weeks after the immediately preceding dose. The phrase“the immediately preceding dose,” as used herein, means, in a sequenceof multiple administrations, the dose of anti-tumor drug which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses.

The methods disclosed herein can comprise administering to a patient anynumber of secondary or subsequent doses of an anti-tumor drug. Forexample, in certain embodiments, only a single second dose isadministered to the patient. In other embodiments, two or more (e.g., 2,3, 4, 5, 6, 7, 8, or more) subsequent doses are administered to thepatient.

In embodiments involving multiple second or subsequent doses, each dosemay be administered at the same frequency or altered frequency as theother doses depending on the relative level of a tumor cell-specificmarker. For example, each subsequent dose may be administered to thepatient 1 to 2 weeks after the immediately preceding dose. The frequencyat which subsequent doses are administered to a patient can vary overthe course of the treatment regimen. The frequency of administration mayalso be adjusted during the course of treatment by a physician dependingon the needs of the individual patient following clinical examination orbased on the relative levels of at least one tumor-responsive biomarkerfollowing administration of each dose.

Accordingly, the level of a tumor cell-specific marker can be monitoredduring multiple administrations of an anti-tumor drug in order determinethe therapeutic efficacy of the anti-tumor drug throughout the course ofadministration. Following each dose, the concentration, level, duration,and/or frequency of anti-tumor drug administration can be altered basedon the level of a tumor-responsive biomarker (e.g., a miRNA marker)following each administration of the anti-tumor drug relative to thelevel of the tumor-responsive biomarker prior to each dose of theanti-tumor drug.

The dosage regimen of an anti-tumor drug can be increased or decreasedin patients having a marker comparison that does not indicatetherapeutic efficacy of an anti-tumor drug. For example, if the markercomparison indicates that the anti-tumor drug was not therapeuticallyeffective, the patient can receive a second or subsequent dose that isthe same or greater than the first dosage of anti-tumor drug. Anincrease in the second or subsequent dose can be a change in the dosageregimen such that the anti-tumor drug is delivered more frequently, in agreater amount, or for a longer time than the previous dose. Forexample, an increase in the second or subsequent dose can be a decreasein the amount of time between doses, or an increase in the amount orconcentration of the dose of anti-tumor drug. For example, the second orsubsequent dose can be increased by a factor of at least 1, at least1.5, at least 2, at least 3, at least 4, at least 5, at least 10, atleast 20 or more. The second or subsequent dose can be administered incombination with another anti-tumor drug or any other therapeuticcomposition.

Likewise, the dosage regimen of patients having a marker comparison thatindicates efficacy of an anti-tumor drug, can be altered or maintainedthe same. In some embodiments, the dosage regimen can be decreased. Forexample, the second dosage can be decreased by a factor of at least 1.5,at least 2, at least 3, at least 4, at least 5, at least 10, at least 20or more. A decrease in the second or subsequent dose can be an increasein the amount of time between doses, or a decrease in the amount orconcentration of the dose. In certain embodiments, the anti-tumor drugis not administered in a second dose. For example, the second dosage ofthe anti-tumor drug can include only different anti-tumor drugs comparedto the first dosage.

In some embodiments, the dosage of an anti-tumor drug can be alteredbased on any medically appropriate reason or based on the relativelevels of multiple tumor-responsive biomarkers before and afteradministration of an anti-tumor drug. Accordingly, the second orsubsequent dosage of anti-tumor drug can be increased or decreased onaverage in a patient population depending on the relative level of atleast one tumor-responsive biomarker before and after administration ofan anti-tumor drug. Due to variations in genetics, patientcharacteristics, environment, and disease subtype between individualpatients, a regime that is effective in one patient may not be effectivein another patient or may be effective to different extents. Within apatient population, at least one patient can be administered a lowersecond dose than the first dose of an anti-tumor drug, at least onepatient can be administered a higher second dose than the first dose ofan anti-tumor drug, and at least one patient can be administered asecond dose that is the same as the first dose of an anti-tumor drug.

Still further provided are methods for assessing a therapeutic efficacyof an anti-tumor drug in a patient. In some embodiments, the methodscomprise (a) obtaining a first baseline sample from the patient'scirculation, wherein the patient has a tumor; (b) administering to thepatient a dose of an anti-tumor drug, wherein at least one tumorcell-specific marker is sequestered within the tumor cell of the patientprior to administration of the anti-tumor drug but is released into thepatient's circulation following administration of the anti-tumor drug;(c) obtaining a second sample from the patient's circulation; (d)measuring an amount of the at least one tumor cell-specific marker (i)in the first baseline sample and (ii) in the second sample, and (e)comparing the amount of the tumor cell-specific marker in the firstbaseline sample with the amount of the tumor cell-specific marker in thesecond sample. An increase in the amount of the at least one tumorcell-specific marker in the second sample relative to the amount of theat least one tumor cell-specific marker in the first baseline sample isindicative of increased tumor cell death in the patient. In certainembodiments the marker is a tumor-responsive biomarker whose levelchanges in response to tumor burden but is not necessarily derived froma tumor. In some embodiments, a biomarker is selected such that adecrease in the amount of the tumor cell-specific marker in the secondsample relative to the amount of the biomarker in the first baselinesample is indicative of increased tumor cell death. The patient can betreated with the anti-tumor drug that increases the at least one tumorcell-specific marker. Alternatively, patients can be treated with theanti-tumor drug that decreases the level of the at least one biomarker.

In some embodiments, the anti-tumor drug is an anti-cancer drug, and thetumor cell-specific marker is a cancer-specific marker. In someembodiments, a single dose of the anti-tumor drug is administered to thepatient. In other embodiments, multiple doses of the anti-tumor drug areadministered to the patient. Accordingly, the level of a biomarker canbe determined following administration of multiple doses of ananti-tumor drug in order to monitor the effectiveness of administrationof the anti-tumor drug throughout administration. In particularembodiments, the biomarker is a tumor-responsive biomarker.

It is also contemplated that embodiments herein can be used to identifyanti-cancer drugs and anti-tumor drugs with high efficacy on particulartypes of cancer or tumors. In particular, a subject, typically a mouseor other like rodent having a pre-determined form of cancer, is testedfor a baseline level of one or more biomolecules associated with thepre-determined form of cancer, e.g., a CD20 marker for lymphoma or amiRNA. The proposed anti-cancer drug for the pre-determined form ofcancer is administered to the subject and the level of the biomoleculedetermined. Increases or decreases of the biomolecule compared tocontrol markers are used to identify anti-cancer drugs with greaterpotency for killing or damaging the pre-determined form of cancer in thesubject. It is contemplated that a plurality of anti-cancer drugs can betested using this method to identify and screen for anti-cancer drugshaving enhanced efficacy for various forms of cancer. In addition,anti-cancer drugs having greater efficacy for one type of cancer overanother type of cancer can also be identified, thereby maximizing cancercell killing and damage, for any one anti-cancer drug or therapeutic.

Anti-tumor drugs selected by the methods disclosed herein can beformulated into a composition for administration to a patient, such as apharmaceutical composition. In certain embodiments, the presentdisclosure provides a pharmaceutical composition comprising one or moreselected anti-tumor drugs as disclosed herein formulated together with apharmaceutically acceptable carrier. The composition may optionallycontain one or more additional pharmaceutically active ingredients, suchas another anti-tumor drug. The pharmaceutical composition can compriseany number of excipients. Excipients that can be used include carriers,surface active agents, thickening or emulsifying agents, solid binders,dispersion or suspension aids, solubilizers, colorants, flavoringagents, coatings, disintegrating agents, lubricants, sweeteners,preservatives, isotonic agents, and combinations thereof. The selectionand use of suitable excipients is taught in Gennaro, ed., Remington: TheScience and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003), the disclosure of which is incorporated herein byreference. The pharmaceutical composition can be formulated to besuitable for intravenous, intramuscular, subcutaneous, parenteral,spinal or epidermal administration (e.g., by injection or infusion).

Also provided herein are methods which measure the levels of certaintumor-specific biomarkers, and tumor-responsive biomarkers such as mRNA,rRNA, microRNAs, cell-free DNA (cfDNA), and other cytosolic, organellaror nuclear macromolecules normally contained within cells, released fromspecific classes of cancer cell, or from normal cells upon developmentof a tumor or in the presence of a tumor, after exposure to ananti-tumor drug compared to release of similar markers from normal cellsafter exposure to the same anti-tumor drug, to determine the extent andratio of tumor cells killed relative to normal cells killed followingthe treatment with the anti-tumor drug, i.e. therapeutic efficacy. Insome embodiments, the release of certain biomarkers such as certainmiRNAs is decreased after exposure to an anti-tumor drug such thattherapeutic efficacy of the anti-tumor drug is found when the level ofextracellular miRNA decreases after administration of the anti-tumordrug. In other embodiments, the marker is a tumor-responsive biomarkerthat is a normal component of the serum, plasma or other body fluid thatchanges in response to tumor burden but is not necessarily derived fromthe tumor. The tumor-responsive biomarkers may be either directly orinversely proportional to the tumor burden.

The biomarkers described herein are useful in methods for identifyingthe efficacy of a therapeutic drug in a cancer subject, i.e. thetherapeutic efficacy or therapeutic index. The level of one or morebiomarkers in a patient that has been treated with an anti-cancer drugcan be determined, and the differential presence of the biomarker can beindicative of the efficacy of the treatment. For example, thedifferential presence of a miRNA before and after administration of ananti-tumor drug to a human subject or non-human animal can be indicativeof the efficacy of the treatment with an anti-tumor drug.

Therapeutic agents (e.g. anti-tumor or anti-cancer drugs) useful incancer treatment for a given subject may be identified using methodsemploying the biomarkers delineated herein. For example, a patient orother subject with cancer may be treated with an anti-cancer drug todetermine the therapeutic index of that anti-cancer drug in thatpatient. The term “efficacy”, “drug efficacy”, “anti-tumor efficacy”, or“therapeutic efficacy” refers to the ability of an anti-tumor drug toslow growth, prevent growth, kill, or damage tumor cells. Thetherapeutic efficacy is determined by measuring the amount of one ormore biomarkers (e.g. a tumor specific biomarker and a non-specificbiomarker or a tumor-responsive biomarker) present in a biologicalsample prior to treatment with the anti-cancer drug and measuring thesame one or more biomarkers after treatment with the anti-cancer drug.The differential presence of the one or more biomarkers in the subjectindicates that the anti-cancer drug may be a useful therapeutic in thatsubject, i.e. the therapeutic index is acceptable for the subject giventhe type and stage of the cancer, etc.

In specific embodiments, the relative level of a tumor-responsivebiomarker marker can correlate to therapeutic efficacy of the anti-tumordrug. Thus, disclosed herein are methods for predicting the therapeuticefficacy of administering an anti-tumor drug by determining the relativelevel of at least one tumor-responsive biomarker marker before and afteradministering a dose of the anti-tumor drug, wherein a change in thelevel of the tumor-responsive biomarker correlates to the ability of ananti-tumor drug to slow growth, prevent growth, kill, or damage tumorcells. Therapeutic efficacy can be determined following a single dose,or following at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10 or more doses of theanti-tumor drug.

In certain embodiments, determining the relative level of atumor-responsive biomarker (e.g., a miRNA marker) before and afteradministration of a putative anti-tumor drug can predict the efficacy ofthe putative anti-tumor drug prior to traditional non-invasivetechniques (e.g., CT scan, MRI scan, PET scan). Accordingly, as usedherein, the term “predictive efficacy” refers to selecting a drug basedon the relative level of a tumor-responsive biomarker before and afteradministration of a putative anti-tumor drug, before efficacy of thedrug could be determined by imaging techniques such as CT scan, MRIscan, and PET scan.

In some embodiments, the anti-tumor drug is an antigen-binding proteinor an antigen-binding fragment thereof. In some embodiments, theantigen-binding protein is a bispecific antigen-binding protein or anantigen-binding fragment thereof. In some embodiments, the anti-tumordrug is a conjugate of an antigen-binding protein and a drug.

Anti-tumor drugs that can be used in the methods disclosed hereininclude, but are not limited to, abitrexate, adriamycin, adrucil,amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine,bicnu, blenoxane, busulfan, bleomycin, camptosar, camptothecins,carboplatin, carmustine, cerubidine, chlorambucil, cisplatin,cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan,dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar,epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine,gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide,ifex, irinotecan, lanvis, leukeran, leustatin, matulane,mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone,mithramycin, mutamycin, myleran, mylosar, navelbine, nipent, novantrone,oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol,plicamycin, procarbazine, purinethol, ralitrexed, taxotere, taxol,teniposide, thioguanine, tomudex, topotecan, valrubicin, velban,vepesid, vinblastine, vindesine, vincristine, vinorelbine, VP-16, andvumon. In certain embodiments, cisplatin is used to treat lung cancertumors, docetaxel is used to treat prostate cancer tumors, andirinotecan is used to treat colon cancer tumors.

Methods for determining tumor-killing efficacy in a patient (e.g., amammal such as a human) can include the step of detecting one or both ofthe presence and amount (or measuring the amount) of one or more tumorcell-specific markers in a sample from a patient. In some embodiments,the presence or amount of the tumor cell-specific marker in the sampleis an indication that the putative anti-tumor drug has efficacy. In someembodiments, methods for determining tumor-killing efficacy can includethe step of determining the level of at least one tumor-responsivebiomarker before and after administration of an anti-tumor drug.Depending on the tumor-responsive biomarker selected, increases ordecreases in the level of tumor-responsive biomarker following drugadministration can be indicative of efficacy.

In some embodiments, the presence of one or more tumor cell-specificmarkers in a sample from a patient is an indication that the putativeanti-tumor drug has efficacy.

In some embodiments, a difference in the amount of one or more tumorcell-specific markers in a sample from a patient as compared to abaseline amount of the marker indicates that the putative anti-tumordrug has efficacy. An increase (e.g., a one-fold, a two-fold, athree-fold, a four-fold, a five-fold, a six-fold, a seven-fold, aneight-fold, a nine-fold, or a ten-fold or more increase) in the amountof a tumor cell-specific marker in the sample as compared to a baselineamount of the marker can be an indication that the putative anti-tumordrug has efficacy. Similarly, a decrease (e.g., a one-fold, a two-fold,a three-fold, a four-fold, a five-fold, a six-fold, a seven-fold, aneight-fold, a nine-fold, or a ten-fold or more decrease) in the amountof a biomarker in the sample as compared to a baseline amount of thebiomarker can be an indication that the putative anti-tumor drug hasefficacy. Likewise, the absence of an increase or the absence of anincrease in the level of a tumor-responsive biomarker in the sample ascompared to a baseline amount of the marker can be an indication thatthe administration of the putative anti-tumor drug does not haveefficacy.

A baseline amount (e.g., an amount or level obtained from a patientprior to treatment with the putative anti-tumor drug or an amount orlevel obtained from one or more subjects prior to treatment with anyanti-tumor drug) can be determined using any of a variety of well-knownmethods. For example, samples from a group of individuals known to havea given cancer can contain, on average, an amount X of a tumorcell-specific marker prior to treatment with any anti-tumor drug,whereas samples from a group of individuals after treatment with a givenanti-tumor drug can contain, on average, an amount of a tumorcell-specific marker that is on average two-fold higher than X ortwo-fold lower than X. In some embodiments, the baseline amount isdetermine based on a tumor-responsive biomarker.

The methods disclosed herein can be performed by a single party or byseparate parties. For example, the party determining the level of atumor-responsive biomarker can be different than the party administeringthe first and subsequent dosages of anti-tumor drug. In someembodiments, the party that determines the level of the tumor-responsivebiomarker is the same party that administers the dosages of theanti-tumor drug.

Sample Collection

Biological samples can be collected from a subject using any acceptableprocedure in the art, for example, by needle aspiration of bodilyfluids, removal of a tissue sample (e.g., biopsy, for example, fineneedle aspiration biopsy, core needle biopsy, or excisional biopsy), andthe like. Typical collection is noninvasive in nature utilizing easilyassessable fluids. However, samples can be obtained from, for example,blood, plasma, serum, cerebrospinal fluid, synovial fluid, lymph,saliva, or urine from a subject. In certain embodiments, the samples areblood samples extracted or drawn from an individual or group ofindividuals by any conventional method. The blood may be drawn from avein or an artery of an individual or group of individuals. Where abiological sample must be stored prior to assay, the biological samplecan be transferred to a glass slide prior to assay or may be frozen forlater preparation or immediately placed in a fixative solution. Thesample extracted from an individual by any means as disclosed above maybe transferred to a tube or container prior to analysis. The containermay be empty, or may comprise a collection media of sorts.

Samples can be collected any time prior to administration of ananti-tumor drug and following administration of an anti-tumor drug. Forexample, samples can be collected immediately before, 6 hours before, 12hours before, 1 day before, 2 days before, 3 days before, 4 days before,5 days before, 6 days before, 8 days before, 10 days before, two weeksbefore, one month before, 1-3 months before, 3-6 months before, 6-12months before, or longer before administration of an anti-tumor drug.Samples can also be collected from the patient immediately after, 6hours after, 12 hours after, 1 day after, 2 days after, 3 days after, 4days after, 5 days after, 10 days after, two weeks after, one monthafter, 1-3 months after, 3-6 months after, or 6-12 months after, orlonger after administration of an anti-tumor drug. In specificembodiments, samples are taken 3 days after administration of ananti-tumor drug.

Markers

Methods provided herein use levels of various biomarkers to achieve thestated goal. In particular, useful markers herein are typicallysequestered within either tumor cells (tumor specific biomarker) orwithin normal cells (control marker). Release of either tumor specificbiomarkers or control markers to the circulation of a patient isindicative that the respective cell type, tumor or normal, has beendamaged or killed. As such, the presence of control markers(particularly when elevated above a baseline level) in the circulationof a patient is an indicator of cell damage and a comparison of levelsreleased by tumor cells to normal cells provides an index or indicationof whether and at what level cancer and normal cells are being killed ordamaged in response to the anti-tumor drug. Non-invasive detection andmeasurement of these markers before and after treatment with a proposedanti-tumor drug can be used to identify, adjust the dose, and determinethe efficacy of any one anti-tumor drug on any one patient and for anyone type of tumor. In some embodiments, the markers are tumor-responsivebiomarkers that change in response to tumor burden but are notnecessarily derived from the tumor.

Biomarkers herein can be a nucleic acid, peptide, protein, lipid,antigen, carbohydrate or proteoglycan, such as DNA (including, forexample, cell-free DNA (cfDNA)) or RNA. The RNA can be mRNA, miRNA,snoRNA, snRNA, rRNAs, tRNAs, siRNA, hnRNA, or shRNA. The cfDNAalterations can include point mutations, DNA hypermethylations,microsatellite instabilities, and losses of heterozygosity. Detecting aplurality of biomarkers can, in some embodiments, provide greatersensitivity or specificity as compared to detecting less than aplurality of biomarkers.

Exemplary tumor cell-specific miRNAs include, but are not limited to,miR-9, miR-15b, miR-15a/miR-16-1, miR-17-3, miR-20a, miR-21, miR-24,miR-25, miR-26a, miR-27, miR-28, miR-30c, miR-92, miR-96-5p, miR-107,miR-122, miR-125a, miR-125a-3p, miR-126, miR-141, miR-145, miR-145-5p,miR-148b, miR-155, miR-182, miR-183-5p, miR-192, miR-194, miR-195,miR-199a, miR-200 family, miR-200a, miR-200b, miR-210, miR-221,miR-221-5p, miR-222, miR-223, miR-298, miR-324-5p, miR-346, miR-375,miR-378, miR-409-3p, miR-423-5p, miR-491, miR-574-3p, miR-622, miR-629,miR-671-3p, miR-1285, let-7c, and let-7e. Exemplary tumor-responsivemiRNA biomarkers also include, but are not limited to, miR-802,miR-30b-3p, miR-510, miR-622, miR-127-3p, miR-373-5p, miR-298,miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p, miR-155-5p,miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p, miR-92a-1-5p,miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p, miR-378a-3p,miR-221-3p, miR-20a5p, miR-93-5p, miR-190, miR-153, miR-26a-2, miR-518c,miR-503, miR-337-3p, miR-518f, miR-370, miR-92a-1, miR-526b, miR-1238,miR-886-3p, miR-887, miR-23a, miR-1267, miR-621, miR-515-3p, miR-424,miR-20b, miR-202, miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p,miR-429, miR-501-3p, miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972,miR-335-3p, miR-16-5p, miR-361-5p, miR-27a-5p, miR-24-3p, miR-1260a,miR-192-5p, miR-548h-5p, miR-122-5p, miR-1208, miR-215, miR-30a-3p,miR-588, miR-10a-3p, miR-21-5p, miR-382-3p, miR-15b-3p, miR-19b-3p,miR-543, miR-1271-5p, miR-106a-5p, miR-106b-5p, miR-520h, miR-181-a2,miR-1468, miR-634, miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a,miR-629, miR-30d-3p, miR-483-5p, miR-708-3p.

In some embodiments, an increase in the level of tumor-responsive miRNAbiomarker in a sample obtained from a patient having cancer followingadministration of an anti-tumor drug is indicative of efficacy of theanti-tumor drug. Exemplary tumor-responsive miRNA biomarkers thatincrease following administration of an effective anti-tumor druginclude, but are not limited to, miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-647,miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof.

In certain embodiments, an increase in the level of lung tumor biomarkermiRNA in a sample obtained from a patient having lung cancer followingadministration of an anti-tumor drug is indicative of efficacy of theanti-tumor drug against lung tumors. Exemplary lung tumor-responsivemiRNA biomarkers that increase following administration of an effectiveanti-tumor drug include, but are not limited to miR-335-3p, miR-16-5p,miR-361-5p, miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p,miR-122-5p, miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p,miR-21-5p, miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p,miR-106a-5p, miR-106b-5p, miR-520h, and a combination thereof.

In certain embodiments, an increase in the level of prostate tumor miRNAbiomarkers in a sample obtained from a patient having prostate cancerfollowing administration of an anti-tumor drug is indicative of efficacyof the anti-tumor drug against prostate tumors. Exemplary prostatetumor-responsive miRNA biomarkers that increase following administrationof an effective anti-tumor drug include, but are not limited tomiR-181-a2, miR-1468, miR-634, miR-647, miR-885-5p, miR-376a, miR-1265,miR-623, miR-15a, miR-629, and a combination thereof.

In certain embodiments, an increase in the level of colon tumor miRNAbiomarkers in a sample obtained from a patient having colon cancerfollowing administration of an anti-tumor drug is indicative of efficacyof the anti-tumor drug against colon tumors. Exemplary colontumor-responsive miRNA biomarkers that increase following administrationof an effective anti-tumor drug include, but are not limited miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof.

In some embodiments, a decrease in the level of tumor-responsive miRNAbiomarkers in a sample obtained from a patient having cancer followingadministration of an anti-tumor drug is indicative of efficacy of theanti-tumor drug. Exemplary tumor-responsive miRNA biomarkers thatdecrease following administration of an effective anti-tumor druginclude, but are not limited to, miR-802, miR-30b-3p, miR-510, miR-622,miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p,miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b,miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p,miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p, miR-190,miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370,miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267,miR-621, miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p, miR-101-5p,miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p, miR-598,miR-206, miR-885-5p, miR-1972, and a combination thereof.

In certain embodiments, a decrease in the level of lung tumor miRNAbiomarkers in a sample obtained from a patient having lung cancerfollowing administration of an anti-tumor drug is indicative of efficacyof the anti-tumor drug against lung tumors. Exemplary lungtumor-responsive miRNA biomarkers that decrease following administrationof an effective anti-tumor drug include, but are not limited to miR-802,miR-30b-3p, miR-510, miR-622, miR-127-3p, miR-373-5p, miR-298,miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p, miR-155-5p,miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p, miR-92a-1-5p,miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p, miR-378a-3p,miR-221-3p, miR-20a 5p, miR-93-5p, and a combination thereof.

In certain embodiments, a decrease in the level of prostate tumor miRNAbiomarkers in a sample obtained from a patient having prostate cancerfollowing administration of an anti-tumor drug is indicative of efficacyof the anti-tumor drug against prostate tumors. Exemplary prostatetumor-responsive miRNA biomarkers that decrease following administrationof an effective anti-tumor drug include, but are not limited to miR-190,miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370,miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267,miR-621, miR-515-3p, miR-424, miR-20b, miR-202, and a combinationthereof.

In certain embodiments, a decrease in the level of colon tumor miRNAbiomarkers in a sample obtained from a patient having colon cancerfollowing administration of an anti-tumor drug is indicative of efficacyof the anti-tumor drug against colon tumors. Exemplary colontumor-responsive miRNA biomarkers that decrease following administrationof an effective anti-tumor drug include, but are not limited tomiR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p,miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972, and a combinationthereof.

Exemplary cancer cell-specific proteins include, but are not limited to,BLK, transglutaminase 4 (TGM4), acid phosphatase (ACPP), CD20,prostate-specific membrane antigen (PSMA), B lymphoid tyrosine kinase(BLK), carcinoembryonic antigen, cytokeratin 19 fragment, cancer antigen125, cancer antigen 15-3, cancer antigen 19-9, BRCA-1, BRCA-2, hCG,thyroglobulin, Hsp27, Hsp70, TGFβ, and alphafetoprotein.

Exemplary cancer cell-specific cell free DNA molecules include DNA withmutations in EGFR, TP53, KRAS, CD98, cathepsin D, and BRAF, epigeneticchanges to glutathione S-transferase P1 and septin 9 genes, andhypermethylation of CDKN2a and APC genes.

Other miRNAs, cell free DNAs, and proteins are known to those skilled inthe art and contemplated herein.

In some embodiments, a nucleic acid sequence (e.g., DNA, RNA, mRNA,miRNA, cell free DNA) can be used as a biomarker, but any relevantpolypeptide sequence encoded thereby can also be used as a biomarker.Accordingly, reference to detection or measurement of a biomarker canrefer to detection or measurement of either or both of a polynucleotideor polypeptide sequence. Biomarkers also include indicators ofepigenetic changes, such as, for example, DNA methylation, mRNAmethylation, histone modification, microRNAs, siRNAs, different spliceforms of RNA, or double stranded RNA.

Exemplary non-specific or control markers include, for example, lactatedehydrogenase (LDH), glutathione reductase (GR), and fatty acid bindingproteins (FABP, including L-FABP and I-FABP), kidney injury molecule-1(Kim-1), S-100B, and neurone specific enolase (NSE). MicroRNA whichindicates acute tissue injury includes miR-208, miR-133, miR-192, miR-1,miR-122, and miR-124. Other non-specific or control markers known tothose skilled in the art are contemplated herein.

Monitoring, measuring, detecting, determining, or observing can be atthe protein or nucleic acid level. Thus, the biomarkers include theseproteins and the genes encoding these proteins. Where detection is atthe protein level, the biomarker protein comprises the full-lengthpolypeptide or any detectable fragment thereof, and can include variantsof these protein sequences. Similarly, where detection is at thenucleotide level, the biomarker nucleic acid includes DNA comprising thefull-length coding sequence, a fragment of the full-length codingsequence, variants of these sequences, for example naturally occurringvariants or splice-variants, or the complement of such a sequence.Biomarker nucleic acids also include RNA, for example, mRNA, comprisingthe full-length sequence encoding the biomarker protein of interest, afragment of the full-length RNA sequence of interest, or variants ofthese sequences. Biomarker proteins and biomarker nucleic acids alsoinclude variants of these sequences. By “fragment” is intended a portionof the polynucleotide or a portion of the amino acid sequence and henceprotein encoded thereby. Polynucleotides that are fragments of abiomarker nucleotide sequence generally comprise at least 10, 15, 20,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 contiguous nucleotides,or up to the number of nucleotides present in a full-length biomarkerpolynucleotide disclosed herein. A fragment of a biomarkerpolynucleotide will generally encode at least 15, 25, 30, 50, 100, 150,200, or 250 contiguous amino acids, or up to the total number of aminoacids present in a full-length biomarker protein of the invention.“Variant” is intended to mean substantially similar sequences.Generally, variants of a particular biomarker of the invention will haveat least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity tothat biomarker as determined by sequence alignment programs known in theart. The protein and corresponding coding sequence for each of thesemarkers is known in the art.

Biomarkers can be classified based on function. For example, biomarkerscan be involved in DNA replication, cell survival/death, ribosomebiogenesis, regulation of signal transduction, and regulation ofprogression through the cell cycle, MAP kinase phosphatase activity,transcription factor activity, cell proliferation, cell-cell signaling,and regulation from a Po1II promoter. Accordingly, it is contemplated,that additional genes and encoded proteins that fall within thesefunctional classifications could be useful biomarkers according to themethods described herein.

Methods provided herein measure the release of microRNAs (and othercytosolic or nuclear macromolecules normally contained within cells)from specific classes of tumor cells compared to release of similarmarkers specific for normal cells, to determine extent and ratio oftumor kill to normal cell kill. Methods are also provided herein thatmeasure the retention of microRNAs from specific classes of tumor cellscompared to the retention of similar markers specific for normal cells,to determine the extent and ratio of tumor kill to normal cell kill.Further provided are methods that measure the general response of amarker to an anti-tumor drug wherein the marker is not necessarily froma tumor.

In some aspects, the tumor is prostate cancer, and the tumorcell-specific marker is a microRNA selected from the group consisting ofmiR-96-5p, miR-183-5p, miR-145-5p, and miR-221-5p. Tumor-responsivemiRNA biomarkers specific for prostate cancer can also include,miR-181-a2, miR-1468, miR-634, miR-647, miR-885-5p, miR-376a, miR-1265,miR-623, miR-15a, miR-629, miR-190, miR-153, miR-26a-2, miR-518c,miR-503, miR-337-3p, miR-518f, miR-370, miR-92a-1, miR-526b, miR-1238,miR-886-3p, miR-887, miR-23a, miR-1267, miR-621, miR-515-3p, miR-424,miR-20b, miR-202, and a combination thereof. In some aspects, the tumorcell-specific marker is prostate-specific membrane antigen (PSMA).

In certain aspects, the tumor is lung cancer and the tumor-responsivebiomarker is a microRNA including miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-802, miR-30b-3p, miR-510, miR-622,miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p,miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b,miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p,miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a 5p, miR-93-5p, and acombination thereof.

In certain aspects, the tumor is colon cancer and the tumor-responsivebiomarker is a microRNA including miR-30d-3p, miR-483-5p, miR-708-3p,miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p,miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972, and a combinationthereof.

In some aspects, the biomarker in the sample is measured and compared tothe level of a control or general marker of cell toxicity such as, e.g.,lactate dehydrogenase or glutathione reductase.

Some biomarkers useful herein represent known genes, the sequences ofwhich are available through public databases known to those of skill inthe art.

In some embodiments, the methods described herein use biomolecules (suchas RNA, microRNA, protein, or DNA) that are normally sequestered withina cancer cell (e.g., cytosolic, nuclear, organellar, membrane-bound,etc.) and are specific to a tumor cell, but are released from the tumorcell when it is damaged or killed by the therapeutic agent. Suchbiomolecules would include, but are not limited to: RNA or DNA orprotein that contain mutations that are specific to the cancer cells;single or multiple species of microRNA that are characteristic of thetumor cell; or RNA, DNA, or proteins that are specific to the celllineage from which the cancer originated. An increase in such abiomarker is an indication of tumor cell killing, and can, in someaspects, be compared to the level of a general marker of cell toxicity(i.e., not specific to the tumor cell), such as lactate dehydrogenase(LDH) or glutathione reductase (GR). In some aspects, the level of sucha biomarker following administration of an anti-tumor drug can becompared to the level of the biomarker prior to administration of theanti-tumor drug in order to determine efficacy of the anti-tumor drug.

In certain embodiments, the methods described herein use biomolecules(such as miRNA) that are normally released by a cancer cell and arespecific to tumor cells, or a specific type of tumor cell, but aresequestered within a cancer cell when it is damaged or killed by thetherapeutic agent. Such biomolecules include single or multiple speciesof microRNA that are characteristic of tumor cells or specific types oftumor cells (e.g., lung cancer or prostate cancer). A decrease in such abiomarker is an indication of tumor cell killing or damage and can, insome aspects, be compared to the level of a general marker of celltoxicity (i.e., not specific to the tumor cell), such as lactatedehydrogenase (LDH) or glutathione reductase (GR). In some aspects, thelevel of such a biomarker following administration of an anti-tumor drugcan be compared to the level of the biomarker prior to administration ofthe anti-tumor drug in order to determine efficacy of the anti-tumordrug. In specific embodiments, the tumor-responsive biomarkers are notreleased by the tumor cell, but are released by the tumor host inresponse to the presence of the tumor. Thus, the tumor-responsivebiomarkers may be either directly or inversely proportional to the tumorburden.

Detection and Quantitation of Biomarkers

Any suitable method can be used to detect (a differential presence of)one or more of the biomarkers described herein. Successful practice ofthe invention can be achieved with one or a combination of methods thatcan detect and/or quantify the biomarkers. These methods include,without limitation, hybridization-based methods including those employedin biochip arrays, mass spectrometry (e.g., laser desorption/ionizationmass spectrometry), fluorescence (e.g., sandwich immunoassay), surfaceplasmon resonance, ellipsometry and atomic force microscopy. For nucleicacid biomarkers, methods for detection and quantitation include PCR,quantitative PCR, northern blot analysis, southern blot analysis, massspectrometry and the like.

Other methods are well known in the art and include but are not limitedto western blots, ELISA, immunoprecipitation, immunofluorescence, flowcytometry, and immunohistochemistry. In particular embodiments,expression of a biomarker is detected on a protein level using, forexample, antibodies that are directed against specific biomarkerproteins. These antibodies can be used in various methods such asWestern blot, ELISA, multiplexing technologies, immunoprecipitation, orimmunohistochemistry techniques. In some embodiments, detection ofcertain markers is accomplished by electrochemiluminescence (ECL).

Methods may further include, by one or more of electrospray ionizationmass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)^(n), matrix-assistedlaser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS), surface-enhanced laser desorption/ionizationtime-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionizationon silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupoletime-of-flight (Q-TOF), atmospheric pressure chemical ionization massspectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)_(n), atmospheric pressurephotoionization mass spectrometry (APPI-MS), APPI-MS/MS, andAPPI-(MS)_(n), quadrupole mass spectrometry, Fourier transform massspectrometry (FTMS), and ion trap mass spectrometry, where n is aninteger greater than zero.

In one embodiment, microarray analysis is used to detect microRNA, knownas microRNA or miRNA expression profiling. The microarray for detectionof microRNA may be a microarray platform, wherein the probes of themicroarray may be comprised of antisense miRNAs or DNA oligonucleotides.In the first case, the target is a labelled sense miRNA sequence, and inthe latter case the miRNA has been reverse transcribed into cDNA andlabelled.

The microarray for detection of microRNA may be a commercially availablearray platform, such as NCode.™. miRNA Microarray Expression Profiling(Invitrogen), miRCURY LNA.™. microRNA Arrays (Exiqon), microRNA Array(Agilent), .mu.Paraflo.®. Microfluidic Biochip Technology (LC Sciences),MicroRNA Profiling Panels (Illumina), Geniom.®. Biochips (Febit Inc.),microRNA Array (Oxford Gene Technology), Custom AdmiRNA.™. profilingservice (Applied Biological Materials Inc.), microRNA Array(Dharmacon-Thermo Scientific), LDA TaqMan analyses (Applied Biosystems),Taqman microRNA Array (Applied Biosystems) or any other commerciallyavailable array.

Microarray analysis may comprise all or a subset of the steps of RNAisolation, RNA amplification, reverse transcription, target labelling,hybridization onto a microarray chip, image analysis and normalization,and subsequent data analysis; each of these steps may be performedaccording to a manufacturers protocol.

It follows, that any of the methods as disclosed herein above e.g. fordiagnosing of an individual may further comprise one or more of thesteps of: [0286] i) isolating miRNA from a sample, [0287] ii) labellingof said miRNA, [0288] iii) hybridizing said labelled miRNA to amicroarray comprising miRNA-specific probes to provide a hybridizationprofile for the sample, [0289] iv) performing data analysis to obtain ameasure of the miRNA expression profile of said sample.

In another embodiment, the microarray for detection of microRNA iscustom made.

A probe or hybridization probe is a fragment of DNA or RNA of variablelength, which is used to detect in DNA or RNA samples the presence ofnucleotide sequences (the target) that are complementary to the sequencein the probe. One example is a sense miRNA sequence in a sample (target)and an antisense miRNA probe. The probe thereby hybridizes tosingle-stranded nucleic acid (DNA or RNA) whose base sequence allowsprobe-target base pairing due to complementarity between the probe andtarget.

To detect hybridization of the probe to its target sequence, the probeor the sample is tagged (or labeled) with a molecular marker. Detectionof sequences with moderate or high similarity depends on how stringentthe hybridization conditions were applied—high stringency, such as highhybridization temperature and low salt in hybridization buffers, permitsonly hybridization between nucleic acid sequences that are highlysimilar, whereas low stringency, such as lower temperature and highsalt, allows hybridization when the sequences are less similar.Hybridization probes used in microarrays refer to nucleotide sequencescovalently attached to an inert surface, such as coated glass slides,and to which a mobile target is hybridized. Depending on the method theprobe may be synthesized via phosphoramidite technology or generated byPCR amplification or cloning (older methods). To design probe sequences,a probe design algorithm may be used to ensure maximum specificity(discerning closely related targets), sensitivity (maximum hybridizationintensities) and normalized melting temperatures for uniformhybridization.

Systems

In another embodiment of the invention, the output from a detectiondevice can subsequently be processed, stored, and further analyzed orassayed using a bio-informatics system. A bio-informatics system mayinclude one or more of the following, without limitation: a computer; aplurality of computers connected to a network; a signal processingtool(s); a pattern recognition tool(s); a tool(s) to control flow ratefor sample preparation, separation, and detection.

The data processing utilizes mathematical foundations. In anotherembodiment of the invention, dynamic programming is used to align aseparation axis with a standard separation profile. Intensities may benormalized, for example, by fitting roughly 90% of the intensity valuesinto a standard spectrum. The data sets can then be fitted usingwavelets designed for separation and mass spectrometer data. In yetanother embodiment of the invention, data processing filters out some ofthe noise and reduces spectrum dimensionality, potentially allowing forpattern recognition.

Following data processing, pattern recognition tools can be utilized toidentify subtle differences between phenotypic states. Patternrecognition tools are based on a combination of statistical and computerscientific approaches, which provide dimensionality reduction. Suchtools are scalable. Data so obtained may be stored on a computerreadable medium.

Kits

In one aspect, the invention provides kits for qualifying cancer statusin a subject, wherein the kits can be used to detect the differentialpresence of the biomarkers described herein. For example, the kits canbe used to detect a differential presence of any combination of thebiomarkers in tumor samples of subjects before and after exposure to atherapeutic drug. The kits of the invention have many applications. Forexample, the kits can be used to monitor efficacy of a therapeutic drugin a cancer subject. The kits can also be used to identify agents usefulin the treatment of cancer.

In specific embodiments, kits of the invention contain an assay for abiomarker, which is optionally isotopically or fluorescently labeled.

The kits of the invention may include instructions, reagents, testingequipment (test tubes, reaction vessels, needles, syringes, etc.),standards for calibration, and/or equipment. Reagents may include acids,bases, oxidizing agents, and marker species. The instructions providedin a kit according to the invention may be directed to suitableoperational parameters in the form of a label or a separate insert.

The kits may also include an adsorbent, wherein the adsorbent retainsone or more biomarkers described herein (polynucleotide or polypeptide),and written instructions for use of the kit for qualification of cancerstatus in a subject. Such a kit could, for example, comprise: (a) asubstrate comprising an adsorbent thereon, wherein the adsorbent issuitable for binding a biomarker, and (b) instructions to detect thebiomarker(s) by contacting a sample with the adsorbent and detecting theproduct(s) retained by the adsorbent. Accordingly, the kit couldcomprise (a) a DNA probe that specifically binds to a biomarker; and (b)a detection reagent. Such a kit could further comprise an eluent (as analternative or in combination with instructions) or instructions formaking an eluent, wherein the combination of the adsorbent and theeluent allows detection of the biomarker using, for example, gas phaseion spectrometry.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

Articles of Manufacture

The methods and materials described herein can be used to, e.g.,determine efficacy of a putative anti-cancer drug and to aid a medicalpractitioner in selecting an appropriate therapy for the subject. To aidin this selection, it may be useful for medicaments used for treating agiven cancer (such as any of the therapies comprising an anti-canceragent described herein) to contain information or appropriate labelsindicating that the medicaments should be prescribed (and/oradministered) to a subject having an having an increase in the level ofa given tumor cell-specific marker following a first administration (ortest administration) of a putative anti-tumor drug. Thus, the disclosurealso features an article of manufacture comprising: a container; and acomposition contained within the container, wherein the compositioncomprises an active agent for treating a given cancer in a subject andwherein the container has a label indicating that the composition is foruse in treating that cancer in a subject if a sample obtained from thesubject after the first administration contains an amount of a tumorcell-specific marker greater than the control amount of that samemarker. The article of manufacture can also contain instructions foradministering the active agent to the subject.

Numbered Embodiments

1. A method of controlling dosage of an anti-tumor drug administered toa patient having cancer, comprising:

-   -   determining a first level of at least one tumor-responsive        biomarker in a first biological sample of the patient, wherein        the first sample is obtained before administering to the patient        a first dose of an anti-tumor drug;    -   determining a second level of the at least one tumor-responsive        biomarker in a subsequent biological sample of the patient,        wherein the subsequent sample is obtained after administering to        the patient the first dose of the anti-tumor drug;    -   wherein thereafter, the patient receives a second dose of the        anti-tumor drug, wherein the dosage regimen of the second dose        depends on whether a decrease or increase in the level of the        tumor-responsive biomarker is identified in the subsequent        biological sample of the patient following administration of the        first dose of the anti-tumor drug.

2. The method of embodiment 1, wherein the at least one tumor-responsivebiomarker includes an miRNA marker selected from the group consistingof: miR-802, miR-30b-3p, miR-510, miR-622, miR-127-3p, miR-373-5p,miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p, miR-155-5p,miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p, miR-92a-1-5p,miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p, miR-378a-3p,miR-221-3p, miR-20a5p, miR-93-5p, miR-190, miR-153, miR-26a-2, miR-518c,miR-503, miR-337-3p, miR-518f, miR-370, miR-92a-1, miR-526b, miR-1238,miR-886-3p, miR-887, miR-23a, miR-1267, miR-621, miR-515-3p, miR-424,miR-20b, miR-202, miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p,miR-429, miR-501-3p, miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972,miR-335-3p, miR-16-5p, miR-361-5p, miR-27a-5p, miR-24-3p, miR-1260a,miR-192-5p, miR-548h-5p, miR-122-5p, miR-1208, miR-215, miR-30a-3p,miR-588, miR-10a-3p, miR-21-5p, miR-382-3p, miR-15b-3p, miR-19b-3p,miR-543, miR-1271-5p, miR-106a-5p, miR-106b-5p, miR-520h, miR-181-a2,miR-1468, miR-634, miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a,miR-629, miR-30d-3p, miR-483-5p, miR-708-3p, and a combination thereof.

3. The method of any one of embodiments 1 or 2, wherein the dosageregimen of the anti-tumor drug is changed if the level of the at leastone tumor-responsive biomarker is not increased.

4. The method of any one of embodiments 1-3, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-647,miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof.

5. The method of any one of embodiments 1-4, wherein the cancer is lungcancer and the at least one tumor-responsive biomarker includes at leastone miRNA marker selected from the group consisting of: miR-335-3p,miR-16-5p, miR-361-5p, miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p,miR-548h-5p, miR-122-5p, miR-1208, miR-215, miR-30a-3p, miR-588,miR-10a-3p, miR-21-5p, miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543,miR-1271-5p, miR-106a-5p, miR-106b-5p, miR-520h, and a combinationthereof.

6. The method of any one of embodiments 1-4, wherein the cancer isprostate cancer and the at least one tumor-responsive biomarker includesat least one miRNA marker selected from the group consisting of:miR-181-a2, miR-1468, miR-634, miR-647, miR-885-5p, miR-376a, miR-1265,miR-623, miR-15a, miR-629, and a combination thereof.

7. The method of any one of embodiments 1-4, wherein the cancer is coloncancer and the at least one tumor-responsive biomarker includes at leastone miRNA marker selected from the group consisting of: miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof.

8. The method of any one of embodiments 1 or 2, wherein the dosageregimen of the anti-tumor drug is changed if the level of the at leastone tumor-responsive biomarker is not decreased.

9. The method of embodiment 1, 2, or 8, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-802, miR-30b-3p, miR-510, miR-622,miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p,miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b,miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p,miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p, miR-190,miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370,miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267,miR-621, miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p, miR-101-5p,miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p, miR-598,miR-206, miR-885-5p, miR-1972, and a combination thereof.

10. The method of embodiment 1, 2, 8, or 9, wherein the cancer is lungcancer, and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of: miR-802,miR-30b-3p, miR-510, miR-622, miR-127-3p, miR-373-5p, miR-298,miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p, miR-155-5p,miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p, miR-92a-1-5p,miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p, miR-378a-3p,miR-221-3p, miR-20a5p, miR-93-5p, and a combination thereof.

11. The method of embodiment 1, 2, 8, or 9, wherein the cancer isprostate cancer, and the at least one tumor-responsive biomarkerincludes at least one miRNA marker selected from the group consistingof: miR-190, miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p,miR-518f, miR-370, miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887,miR-23a, miR-1267, miR-621, miR-515-3p, miR-424, miR-20b, miR-202, and acombination thereof.

12. The method of embodiment 1, 2, 8, or 9, wherein the cancer is coloncancer, and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of: miR-21-3p,miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p,miR-598, miR-206, miR-885-5p, miR-1972, and a combination thereof.

13. The method of any one of embodiments 1-12, further comprising:

-   -   determining the amount of a control marker before and after        administering to the patients the first dose of the anti-tumor        drug, wherein the second dose of the anti-tumor drug is adjusted        according to the change in the level of the tumor-responsive        biomarker compared to the change in the level of the control        marker in the determining step,    -   wherein the control marker is different from the        tumor-responsive biomarker.

14. The method of any one of embodiments 1-13, wherein the tumor isselected from the group consisting of non-Hodgkin's lymphoma, chroniclymphocytic leukemia, multiple myeloma, B cell lymphoma, high-grade Bcell lymphoma, intermediate-grade B cell lymphoma, low-grade B celllymphoma, B cell acute lymphoblastic leukemia, Hodgkin's disease,plasmacytoma, follicular lymphoma, follicular small cleaved lymphoma,follicular large cell lymphoma, follicular mixed small cleaved lymphoma,diffuse small cleaved cell lymphoma, diffuse small lymphocytic lymphoma,prolymphocytic leukemia, lymphoplasmacytic lymphoma, marginal zonelymphoma, mucosal associated lymphoid tissue lymphoma, monocytoid B celllymphoma, splenic lymphoma, hairy cell leukemia, diffuse large celllymphoma, mediastinal large B cell lymphoma, lymphomatoidgranulomatosis, intravascular lymphomatosis, diffuse mixed celllymphoma, diffuse large cell lymphoma, immunoblastic lymphoma, Burkitt'slymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, mantlecell lymphoma, heavy chain disease, lung carcinoma, breast carcinoma,ovarian carcinoma, skin carcinoma, colon carcinoma, urinary bladdercarcinoma, liver carcinoma, gastric carcinoma, prostate cancer, renalcell carcinoma, nasopharyngeal carcinoma, squamous cell carcinoma,thyroid papillary carcinoma, cervical carcinoma, and sarcomas.

15. The method of any one of embodiments 1-14, wherein the firstbiological sample and the second biological sample are fluid samplesobtained from blood, plasma, serum, cerebrospinal fluid, synovial fluid,lymph, saliva, or urine of the patient.

16. The method of any one of embodiments 1-15, wherein the secondbiological sample is obtained from the patient immediately after, 6hours after, 12 hours after, 1 day after, 2 days after, 3 days after, 4days after, 5 days after, 10 days after, two weeks after, one monthafter, 1-3 months after, 3-6 months after, or 6-12 months afteradministration of the first dose of the anti-tumor drug.

17. The method of any one of embodiments 1-16, wherein the therapeuticefficacy of the anti-tumor drug increases following administration ofthe second dose of the anti-tumor drug.

18. The method of any one of embodiments 1-17, wherein the anti-tumordrug includes cisplatin, docetaxel, or irinotecan.

19. The method of any one of embodiments 1-18, wherein the second doseof the anti-tumor drug is administered along with a different anti-tumordrug.

20. A method for administering at least one anti-tumor drug in twoseparate doses to a patient having cancer, comprising:

-   -   (a) obtaining a first biological sample from the patient;    -   (b) determining a baseline level of at least one        tumor-responsive biomarker in the first biological sample;    -   (c) administering the first dose of an anti-tumor drug to the        patient;    -   (d) obtaining a second biological sample from the patient;    -   (e) determining a first level of the at least one        tumor-responsive biomarker in the second biological sample;    -   (f) comparing the baseline level and the first level of        tumor-responsive biomarker to identify if the patient has a        decrease or increase in the level of the at least one        tumor-responsive biomarker; and    -   (g) administering the second dose of the anti-tumor drug to the        patient,    -   wherein the dosage regimen of the second dose is changed if the        patient has a decrease or increase in the level of the at least        one tumor-responsive biomarker.

21. The method of embodiment 20, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-802, miR-30b-3p, miR-510, miR-622,miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p,miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b,miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p,miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p, miR-190,miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370,miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267,miR-621, miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p, miR-101-5p,miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p, miR-598,miR-206, miR-885-5p, miR-1972, miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-885-5p,miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p, miR-483-5p,miR-708-3p, and a combination thereof.

22. The method of embodiment 20 or 21, wherein the dosage regimen of theanti-tumor drug is changed if the level of the at least onetumor-responsive biomarker is not increased.

23. The method of any one of embodiments 20-22, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-647,miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof.

24. The method of any one of embodiments 20-23, wherein the cancer islung cancer and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of:miR-335-3p, miR-16-5p, miR-361-5p, miR-27a-5p, miR-24-3p, miR-1260a,miR-192-5p, miR-548h-5p, miR-122-5p, miR-1208, miR-215, miR-30a-3p,miR-588, miR-10a-3p, miR-21-5p, miR-382-3p, miR-15b-3p, miR-19b-3p,miR-543, miR-1271-5p, miR-106a-5p, miR-106b-5p, miR-520h, and acombination thereof.

25. The method of any one of embodiments 20-23, wherein the cancer isprostate cancer and the at least one tumor-responsive biomarker includesat least one miRNA marker selected from the group consisting of:miR-181-a2, miR-1468, miR-634, miR-647, miR-885-5p, miR-376a, miR-1265,miR-623, miR-15a, miR-629, and a combination thereof.

26. The method of any one of embodiments 20-23, wherein the cancer iscolon cancer and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of:miR-30d-3p, miR-483-5p, miR-708-3p, and a combination thereof.

27. The method of embodiment 20 or 21, wherein the dosage regimen of theanti-tumor drug is changed if the level of the at least onetumor-responsive biomarker is not decreased.

28. The method of embodiment 20, 21, or 27, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-802, miR-30b-3p, miR-510, miR-622,miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p,miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b,miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p,miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p, miR-190,miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370,miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267,miR-621, miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p, miR-101-5p,miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p, miR-598,miR-206, miR-885-5p, miR-1972, and a combination thereof.

29. The method of embodiment 20, 21, 27, or 28, wherein the cancer islung cancer, and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of: miR-802,miR-30b-3p, miR-510, miR-622, miR-127-3p, miR-373-5p, miR-298,miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p, miR-155-5p,miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p, miR-92a-1-5p,miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p, miR-378a-3p,miR-221-3p, miR-20a5p, miR-93-5p, and a combination thereof.

30. The method of embodiment 20, 21, 27, or 28, wherein the cancer isprostate cancer, and the at least one tumor-responsive biomarkerincludes at least miRNA marker selected from the group consisting of:miR-190, miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f,miR-370, miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a,miR-1267, miR-621, miR-515-3p, miR-424, miR-20b, miR-202, and acombination thereof.

31. The method of embodiment 20, 21, 27, or 28, wherein the cancer iscolon cancer, and the at least one tumor-responsive biomarker includesat least one miRNA marker selected from the group consisting of:miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p,miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972, and a combinationthereof.

32. The method of any one of embodiments 20-31, wherein the tumor isselected from the group consisting of non-Hodgkin's lymphoma, chroniclymphocytic leukemia, multiple myeloma, B cell lymphoma, high-grade Bcell lymphoma, intermediate-grade B cell lymphoma, low-grade B celllymphoma, B cell acute lymphoblastic leukemia, Hodgkin's disease,plasmacytoma, follicular lymphoma, follicular small cleaved lymphoma,follicular large cell lymphoma, follicular mixed small cleaved lymphoma,diffuse small cleaved cell lymphoma, diffuse small lymphocytic lymphoma,prolymphocytic leukemia, lymphoplasmacytic lymphoma, marginal zonelymphoma, mucosal associated lymphoid tissue lymphoma, monocytoid B celllymphoma, splenic lymphoma, hairy cell leukemia, diffuse large celllymphoma, mediastinal large B cell lymphoma, lymphomatoidgranulomatosis, intravascular lymphomatosis, diffuse mixed celllymphoma, diffuse large cell lymphoma, immunoblastic lymphoma, Burkitt'slymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, mantlecell lymphoma, heavy chain disease, lung carcinoma, breast carcinoma,ovarian carcinoma, skin carcinoma, colon carcinoma, urinary bladdercarcinoma, liver carcinoma, gastric carcinoma, prostate cancer, renalcell carcinoma, nasopharyngeal carcinoma, squamous cell carcinoma,thyroid papillary carcinoma, cervical carcinoma, and sarcomas.

33. The method of any one of embodiments 20-32, wherein the firstbiological sample and the second biological sample are fluid samplesobtained from blood, plasma, serum, cerebrospinal fluid, synovial fluid,lymph, saliva, or urine of the patient.

34. The method of any one of embodiments 20-33, wherein the secondbiological sample is obtained from the patient immediately after, 6hours after, 12 hours after, 1 day after, 2 days after, 3 days after, 4days after, 5 days after, 10 days after, two weeks after, one monthafter, 1-3 months after, 3-6 months after, or 6-12 months afteradministration of the first dose of the anti-tumor drug.

35. The method of any one of embodiments 20-34, wherein the therapeuticefficacy of the anti-tumor drug increases following administration ofthe second dose of the anti-tumor drug.

36. The method of any one of embodiments 20-35, wherein the anti-tumordrug includes cisplatin, docetaxel, or irinotecan.

37. The method of any one of embodiments 20-36, wherein the second doseof the anti-tumor drug is administered along with a different anti-tumordrug.

38. A method of selecting at least one anti-tumor drug, the methodcomprising:

-   -   determining the level of at least one tumor-responsive biomarker        before and after administering a candidate anti-tumor drug to a        host non-human animal having a tumor; and    -   selecting an anti-tumor drug if the candidate anti-tumor drug        increases or decreases the level of the at least one        tumor-responsive biomarker in the host non-human animal.

39. The method of embodiment 38, wherein the at least one selectedanti-tumor drug is formulated into a composition.

40. The method of embodiment 38, further comprising:

-   -   (a) providing a host non-human animal having a tumor;    -   (b) obtaining a first biological sample from the host non-human        animal;    -   (c) determining the baseline level of at least one        tumor-responsive biomarker in the first biological sample;    -   (d) administering a candidate anti-tumor drug to the host        non-human animal;    -   (e) obtaining a second biological sample from the host non-human        animal;    -   (f) determining a first level of the at least one        tumor-responsive biomarker in the second biological sample;    -   (g) comparing the baseline level and the first level of the        tumor-specific miRNA marker to identify if the patient has a        decrease or increase in the level of the at least one        tumor-responsive biomarker; and    -   (h) selecting the candidate anti-tumor drug as an anti-tumor        drug if administration of the candidate anti-tumor drug leads to        the increase or decrease in the level of the tumor-responsive        biomarker in the host non-human animal.

41. The method of any one of embodiments 38-40, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-802, miR-30b-3p, miR-510, miR-622,miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p,miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p, miR-190b,miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p, miR-20a-5p,miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p, miR-190,miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f, miR-370,miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a, miR-1267,miR-621, miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p, miR-101-5p,miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p, miR-598,miR-206, miR-885-5p, miR-1972, miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-885-5p,miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p, miR-483-5p,miR-708-3p, and a combination thereof.

42. The method of any one of embodiments 38-41, wherein the candidateanti-tumor drug is selected if the level of the at least onetumor-responsive biomarker is increased.

43. The method of any one of embodiments 38-42, wherein the at least onetumor-responsive biomarker includes at least one miRNA marker selectedfrom the group consisting of: miR-335-3p, miR-16-5p, miR-361-5p,miR-27a-5p, miR-24-3p, miR-1260a, miR-192-5p, miR-548h-5p, miR-122-5p,miR-1208, miR-215, miR-30a-3p, miR-588, miR-10a-3p, miR-21-5p,miR-382-3p, miR-15b-3p, miR-19b-3p, miR-543, miR-1271-5p, miR-106a-5p,miR-106b-5p, miR-520h, miR-181-a2, miR-1468, miR-634, miR-647,miR-885-5p, miR-376a, miR-1265, miR-623, miR-15a, miR-629, miR-30d-3p,miR-483-5p, miR-708-3p, and a combination thereof.

44. The method of any one of embodiments 38-42, wherein the cancer islung cancer and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of:miR-335-3p, miR-16-5p, miR-361-5p, miR-27a-5p, miR-24-3p, miR-1260a,miR-192-5p, miR-548h-5p, miR-122-5p, miR-1208, miR-215, miR-30a-3p,miR-588, miR-10a-3p, miR-21-5p, miR-382-3p, miR-15b-3p, miR-19b-3p,miR-543, miR-1271-5p, miR-106a-5p, miR-106b-5p, miR-520h, and acombination thereof.

45. The method of any one of embodiments 38-42, wherein the cancer isprostate cancer and the at least one tumor-responsive biomarker includesat least one miRNA marker selected from the group consisting of:miR-181-a2, miR-1468, miR-634, miR-647, miR-885-5p, miR-376a, miR-1265,miR-623, miR-15a, miR-629, and a combination thereof.

46. The method of any one of embodiments 38-42, wherein the cancer iscolon cancer and the at least one tumor-responsive biomarker includes atleast one miRNA marker selected from the group consisting of:miR-30d-3p, miR-483-5p, miR-708-3p, and a combination thereof.

47. The method of any one of embodiments 38-41, wherein the candidateanti-tumor drug is selected if the level of the at least onetumor-responsive biomarker is decreased.

48. The method of any one of embodiments 38-41 or 47, wherein the atleast one tumor-responsive biomarker includes at least one miRNA markerselected from the group consisting of: miR-802, miR-30b-3p, miR-510,miR-622, miR-127-3p, miR-373-5p, miR-298, miR-302b-3p, miR-367-3p,miR-181b-5p, miR-518a-3p, miR-155-5p, miR-214-3p, miR-329, let-7f-5p,miR-190b, miR-503-5p, miR-92a-1-5p, miR-647, miR-153, miR-93-5p,miR-20a-5p, miR-221-3p, miR-378a-3p, miR-221-3p, miR-20a5p, miR-93-5p,miR-190, miR-153, miR-26a-2, miR-518c, miR-503, miR-337-3p, miR-518f,miR-370, miR-92a-1, miR-526b, miR-1238, miR-886-3p, miR-887, miR-23a,miR-1267, miR-621, miR-515-3p, miR-424, miR-20b, miR-202, miR-21-3p,miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p, miR-509-3p,miR-598, miR-206, miR-885-5p, miR-1972, and a combination thereof.

49. The method of any one of embodiments 38-41, 47, or 48, wherein thecancer is lung cancer, and the at least one tumor-responsive biomarkerincludes at least one miRNA marker selected from the group consistingof: miR-802, miR-30b-3p, miR-510, miR-622, miR-127-3p, miR-373-5p,miR-298, miR-302b-3p, miR-367-3p, miR-181b-5p, miR-518a-3p, miR-155-5p,miR-214-3p, miR-329, let-7f-5p, miR-190b, miR-503-5p, miR-92a-1-5p,miR-647, miR-153, miR-93-5p, miR-20a-5p, miR-221-3p, miR-378a-3p,miR-221-3p, miR-20a5p, miR-93-5p, and a combination thereof.

50. The method of any one of embodiments 38-41, 47, or 48, wherein thecancer is prostate cancer, and the at least one tumor-responsivebiomarker includes at least one miRNA marker selected from the groupconsisting of: miR-190, miR-153, miR-26a-2, miR-518c, miR-503,miR-337-3p, miR-518f, miR-370, miR-92a-1, miR-526b, miR-1238,miR-886-3p, miR-887, miR-23a, miR-1267, miR-621, miR-515-3p, miR-424,miR-20b, miR-202, and a combination thereof.

51. The method of any one of embodiments 38-41, 47, or 48, wherein thecancer is colon cancer, and the at least one tumor-responsive biomarkerincludes at least one miRNA marker selected from the group consistingof: miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p,miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972, and a combinationthereof.

52. The method of any one of embodiments 38-51, further comprisingintroducing a tumor cell, a tumor tissue, or a tumor organ into the hostnon-human animal to provide the host non-human animal having the tumor.

53. The method of any one of embodiments 38-52, wherein the tumor isderived from a human patient having a cancer, and wherein the non-humananimal is a rodent.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1

The tumor cell being targeted is a B-cell lymphoma that expresses thecell surface protein CD20 (also called MS4A1). Drugs that targetCD20-expressing cells, such as conventional antibodies, enhancedantibodies, or bispecific antibodies that engage other effector cellssuch as T-cells, cause damage to the B-cell lymphoma tumor cells andthereby release specific biomolecules that can be detected in thecirculation.

In this example, mice bearing Raji cell tumors (a B-cell lymphoma tumorline) are treated with a bispecific antibody containing a CD20 bindingarm and a CD3 binding arm (which engages effector T-cells and activateskilling of the Raji cells). As a result of treatment of the mice withthis CD20xCD3 bispecific reagent, increased levels of biomolecules thatare specific to the Raji cells are found in the circulation of the mice.These biomolecules include cytosolic proteins such as B lymphoidtyrosine kinase (BLK) and DNA such as the specifically rearrangedimmunoglobulin gene from the Raji cell.

Example 2

The tumor cell being targeted is a prostate cancer cell that expressesprostate specific cell surface proteins such as FOLH1 (also calledPSMA), Steap1, or Steap2. Drugs that target FOLH1-expressing cells, suchas conventional antibodies, enhanced antibodies, or antibody drugconjugates that are linked to toxins, cause damage to the prostate tumorcells and thereby release specific biomolecules that can be detected inthe circulation.

In this example, mice bearing LnCAP tumors (a prostate cancer tumorline) are treated with an antibody drug conjugate (ADC) consisting of anantibody directed to FOLH1 conjugated via a non-cleavable linker to amaytansinoid toxic drug. As a result of treatment of the mice with thisFOLH1-directed ADC, increased levels of biomolecules that are specificto the LnCAP cells are found in the circulation of the mice. Thesebiomolecules include cytosolic proteins such as transglutaminase 4(TGM4) and acid phosphatase (ACPP), and DNA such as the TMPRSS2-ERG genefusion that is common in prostate cancer. Further, treatment alsochanges the pattern of certain microRNA's that are found in thecirculation; these microRNA's include miR's 96-5p, 183-5p, 145-5p, and221-5p.

Example 3: Method of miRNA Profiling

Plasma samples (50 uL) were stored at −80° C. after receiving them. Thesamples were thawed on ice, and total RNA was isolated with the miRCURYRNA Isolation Kit for Biofluids (Exiqon) according to the suppliedprotocol (1 uL of Exiqon spike-in UniRT RNA mix was added to each samplebefore RNA isolation). RNA was eluted into 50 uL nuclease-free water,and either processed directly for cDNA synthesis of stored at −80° C.until that time. RNA from each sample was used to create cDNA in 40 uLreaction volume with the Universal cDNA Synthesis Kit II (Exiqon)according to the supplied protocol. RNA concentration for plasma samplesis not quantitated; 8 uL each RNA sample is used without adjustingconcentration. The 40 uL of cDNA per sample was then used to set-up qPCRdirectly or stored at −20° C. until use. The 40 uL cDNA was diluted into4 mL of nuclease-free water, and then mixed with 4 mL 2X ExiLENT SYBRGreen master mix (Exiqon) to create 8 mL qPCR mix, according to protocolsupplied with miRNome Panels (Exiqon). The 8 ml master mix was thenpipetted into the two 384-well plates (Exiqon microRNA Ready-to-Use PCR,Human panel I+II, V3.M), 10 ul per well according to the suppliedprotocol. The plates were briefly centrifuged, sealed, and run on theABI Viia7 real time PCR thermocycler, using the PCR template programsupplied by Exiqon. Results were exported as text files and thenimported into the GenEx software (Exiqon) for analysis. Datapre-processing and analysis steps were performed according to therecommendations of the Exiqon Data Analysis Guide. Briefly, biologicalreplicate were grouped, outliers were automatically detected andremoved, Ct values higher than 37 were considered background andremoved, missing data was filled by the GenEx software with an imputedvalue based on group mean. Cts for each sample were normalized to theglobal mean of all expressed miRNAs with a Ct<34. Various analyses couldbe done in GenEx, including clustering analysis and t-tests betweendifferent groups of samples to determine miRNAs significantly alteredbetween groups.

Example 4: Drug efficacy of COLO 205 Tumor Treated with Irinotecan

Immune compromised mice were injected with COLO 205 human colorectalcancer cells and tumors were allowed to develop, after which the micewere treated with either irinotecan or vehicle for 14 days. In a secondcontrol group, normal mice maintained in parallel but not bearing tumorswere treated with either irinotecan or vehicle. A third control groupconsisted of tumor-bearing mice or normal, tumor-free mice that did notreceive any treatment. During the course of drug treatment, blood plasmasamples were taken from all mice at regular intervals from 3 to 14 days.RNA was prepared from the plasma samples and analyzed for miRNA contentby a panel of quantitative PCR assays for approximately 700 miRNAs. Therelative quantities of each miRNA assayed were normalized to the globalmean in each sample and then compared between the paired groups: drugversus vehicle treatment in tumor-bearing mice; drug versus vehicle intumor-free mice; and tumor bearing versus tumor-free mice. Samples weretaken from six replicate mice for each time point in each of thecategories. MicroRNAs whose mean values for the six replicates differedby a factor of at least two between the comparative categories and had aP value (Student's T-test) less than 0.05 were examined further.

After 14 days of drug treatment there was a significantly lower tumorburden in the drug treated mice than in the vehicle treated controlgroup. Comparing the plasma miRNA profiles between the drug-treated andvehicle control group at the 14 day time point revealed many different,with some miRNAs more abundant and others less abundant in the drugtreated mice compared with the controls. To eliminate those miRNAdifferences that were the result of drug treatment alone, we examinedthe miRNA profiles in the irinotecan treated normal, tumor-free micecompared with the same type of mice treated with vehicle. All miRNAsthat showed the same directional differences (e.g. higher in the drugtreated group than in the vehicle treated controls) that we observed inthe tumor bearing experimental group were eliminated from considerationas part of a drug efficacy signature.

The miRNA profiles were examined in the plasma samples from thetumor-bearing mice treated with drug or vehicle for only three days, atime in the course of drug treatment where no significant difference intumor burden was seen. We looked for miRNAs whose pattern of detectionmatched that in the mice treated with drug for 14 days. The result wasthe 14 miRNAs shown in Table 1. The pattern of quantification for thesemiRNAs—higher or lower in the drug treated mice compared to the vehicletreated controls—matched miRNAs from the 14 day time point. We foundthree miRNAs, miR-30d-3p, miR-483-5p, and miR-708-3p, that were elevatedin the drug treated tumor bearing mice compared with the vehiclecontrols and 11 miRNAs, miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p,miR-429, miR-501-3p, miR-509-3p, miR-598, miR-206, miR-885-5p, andmiR-1972, that were lower in the drug treated mice compared with thecontrols. These 14 miRNAs form a signature of plasma miRNA content thatcorrelates with drug efficacy late in the course of drug treatment andpredicts efficacy early in the drug treatment regimen before any effectsof irinotecan on tumor burden could be detected.

To better understand and validate the drug efficacy signature, we lookedat the content of the 14 signature miRNAs in untreated tumor bearingmice compared to normal tumor-free controls. The 11 miRNAs listed ashaving lower plasma contents in the drug treated mice compared with thecontrols (column 2 of Table 1) actually had the opposite pattern in theuntreated tumor-bearing mice—they were detected in the tumor-bearingmice but were below the limit of detection in the normal controls.Detection of these miRNAs in plasma correlates with tumor load.Irinotecan treatment reversed this pattern—the 11 miRNAs went fromdetectable in the vehicle treated mice to undetectable in the drugtreated mice, reflecting the efficacy of irinotecan at reducing tumorload. The three miRNAs listed as having higher plasma contents in thedrug treated mice compared with the controls (left side of Table 1)showed a mirror pattern—they were detected in normal mice but wereeither reduced (miR-483-5p) or not detected at all (miR-30d-3p andmiR-708-3p) in tumor-bearing mice, while irinotecan treatment in thetumor-bearing mice reversed this pattern; they either went fromundetected to detected or, in the case of miR-483-5p, increased.

TABLE 1 Drug efficacy signature for COLO 205 tumor treated withirinotecan miRNAs different upon drug treatment* Higher Lower miR-30d-3pmiR-21-3p miR-483-5p miR-101-5p miR-708-3p miR-122-3p miR-197-3p miR-429miR-501-3p miR-509-3p miR-598 miR-206 miR-885-5p miR-1972 *Relative to avehicle-treated control

Thus, the 14 miRNAs in the COLO 205 drug efficacy signature serve assensitive early predictors of irinotecan anti-tumor drug efficacy. Atthe same time they form a diagnostic signature of tumor load. Themethods described herein to derive the drug efficacy signature for theCOLO 205 tumor could be applied to other drug treatments and other humanxenograft tumor models as well as genetically induced tumors. It wouldalso be possible to apply the same methods to any disease situation forwhich effective therapies exist or are being developed.

While this invention has been described with an emphasis upon typicalembodiments, it will be understood by those of ordinary skill in the artthat variations of the typical embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

1-53. (canceled)
 54. A method of controlling dosage of an anti-tumordrug administered to human patients having a colon cancer, comprising:determining a first level of a panel of tumor-responsive biomarkerscomprising miR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p, miR-429,miR-501-3p, miR-509-3p, miR-598, miR-206, miR-885-5p, miR-1972,miR-30d-3p, miR-483-5p, and miR-708-3p in first biological samples ofthe patients, wherein the first samples are obtained beforeadministering to the patients a first dose of an anti-tumor drug;determining a second level of the panel of tumor-responsive biomarkersin subsequent biological samples of the patients, wherein the subsequentsamples are obtained after administering to the patients the first doseof the anti-tumor drug; wherein increases in each of biomarkersmiR-30d-3p, miR-483-5p, and miR-708-3p and decreases in each ofmiR-21-3p, miR-101-5p, miR-122-3p, miR-197-3p, miR-429, miR-501-3p,miR-509-3p, miR-598, miR-206, miR-885-5p, and miR-1972 indicate afavorable response to treatment thereafter, administering differentregimens of the drug to different patients depending on the response totreatment indicated by the biomarkers in the patients.
 55. The method ofclaim 54, further comprising: determining the amount of a control markerin the patients before and after administering to the patients the firstdose of the anti-tumor drug, wherein the different regimens of the drugadministered to the different patients are adjusted according to thechange in the level of the tumor-responsive biomarkers in the patientscompared to the change in the level of the control marker in thepatients in the determining step, wherein the control marker isdifferent from the tumor-responsive biomarkers.
 56. The method of claim54, wherein the first biological samples and the subsequent biologicalsamples are fluid samples obtained from blood, plasma, serum,cerebrospinal fluid, synovial fluid, lymph, saliva, or urine of thepatients.
 57. The method of claim 54, wherein the subsequent biologicalsamples are obtained from the patients immediately after, 6 hours after,12 hours after, 1 day after, 2 days after, 3 days after, 4 days after, 5days after, 10 days after, two weeks after, one month after, 1-3 monthsafter, 3-6 months after, or 6-12 months after administration of thefirst dose of the anti-tumor drug.
 58. The method of claim 54, whereinthe anti-tumor drug is irinotecan.
 59. The method of claim 54, whereinthe different regimens of the anti-tumor drug administered to thedifferent patients are administered to the different patients along witha different anti-tumor drug.
 60. The method of claim 54, wherein thedetermining of the first level and the second level of the panel oftumor-responsive biomarkers is by microarray analysis.